Glucagon-like-peptide-2 (GLP-2) analogues

ABSTRACT

GLP-2 analogues are disclosed which comprise one of more substitutions as compared to [hGly2]GLP-2 and which improved biological activity in vivo and/or improved chemical stability, e.g. as assessed in in vitro stability assays. More particularly, preferred GLP-2 analogues disclosed herein comprise substitutions at one or more of positions 8, 16, 24 and/or 28 of the wild-type GLP-2 sequence, optionally in combination with further substitutions at position 2 (as mentioned in the introduction) and one or more of positions 3, 5, 7, 10 and 11, and/or a deletion of one or more of amino acids 31 to 33 and/or the addition of a N-terminal or C-terminal stabilizing peptide sequence. The analogues are particularly useful for the prophylaxis or treatment of stomach and bowel-related disorders and for ameliorating side effects of chemotherapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit from U.S. Provisional Application No.60/678,066, filed May 4, 2005, which is hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to glucagon-like-peptide-2 (GLP-2)analogues and their medical use, for example in the prophylaxis ortreatment of stomach and bowel-related disorders and for amelioratingside effects of chemotherapy and radiation therapy.

BACKGROUND OF THE INVENTION

GLP-2 is a 33-amino-acid peptide released from the posttranslationalprocessing of proglucagon in the enteroendocrine L cells of theintestine and in specific regions of the brainstem. It is co-secretedtogether with glucagon-like peptide 1 (GLP-1), oxyntomodulin andglicentin, in response to nutrient ingestion.

GLP-2 induces significant growth of the small intestinal mucosalepithelium via the stimulation of stem cell proliferation in the cryptsand inhibition of apoptosis on the villi (Drucker et al. Proc Natl AcadSci USA. 1996, 93:7911-6). GLP-2 also inhibits gastric emptying andgastric acid secretion (Wojdemann et al. J Clin Endocrinol Metab. 1999,84:2513-7), enhances intestinal barrier function (Benjamin et al. Gut.2000, 47:112-9.), stimulates intestinal hexose transport via theupregulation of glucose transporters (Cheeseman, Am J. Physiol. 1997,R1965-71), and increases intestinal blood flow (Guan et al.Gastroenterology. 2003, 125, 136-47).

GLP-2 binds to a single G protein-coupled receptor belonging to theclass II glucagon secretin family (1). The GLP-2 receptor has only beenlocalized in the small intestine, colon and stomach, sites that areknown to be responsive to GLP-2 (Yusta et al. Gastroenterology. 2000,119: 744-55). However, the target cell for GLP-2 receptor stimulation inthe gastrointestinal tract remanins unclear and the downstreamintracellular mediators coupled to the GLP-2 receptor are poorlyunderstood.

The demonstrated specific and beneficial effects of GLP-2 in the smallintestine has raised much interest as to the use of GLP-2 in thetreatment of intestinal disease or injury (Sinclair and Drucker,Physiology 2005: 357-65). Furthermore GLP-2 has been shown to prevent orreduce mucosal epithelial damage in a wide number of preclinical modelsof gut injury, including chemotherapy-induced mucositis,ischemia-reperfusion injury, dextran sulfate-induced colitis and geneticmodels of inflammatory bowel disease (Sinclair and Drucker, Physiology2005:357-65).

GLP-2 is secreted as a 33 amino acid peptide with the following sequenceH-His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-OH(SEQ ID NO:50). It is rapidly cleaved at the Alanine (A) in position 2of the NH₂ terminus to the inactive human GLP-2 (3-33) by the enzyme DPPIV. This rapid enzymatic degradation of GLP-2(1-33), in addition torenal clearance result in a half life of about 7 minutes for the peptide(Tavares et al., Am. J. Physiol. Endocrinol. Metab. 278:E134-E139,2000).

In U.S. Pat. No. 5,994,500 (Drucker et al.) describes antagonists of theGLP-2 and their effects on the growth of gastrointestinal tissue. It issuggested that the antagonists are formulated as pharmaceuticals to beused in the treatment of hyperplasia or to induce hypoplasia. In U.S.Pat. No. 5,994,500 the structure of mammalian GLP-2 has been altered bymutations, such as substitutions and deletions.

U.S. Pat. Nos. 6,184,208, 5,789,379, and 6,184,201 disclose GLP-2analogues and their medical uses. The analogues are all obtained bysubstitutions and/or deletions of the human GLP-2.

DaCambra et al. (Biochemistry 2000, 39, 8888-8894) describe thestructural determinants for activity of GLP-2. Examples of suchdeterminants are Phe6 and Thr5, which are referred to as crucial forGLP-2 receptor binding and activation.

In WO 97/39031 the GLP-2 analogue, [Gly2]GLP-2 (SEQ ID NO:54) isdisclosed. Here the alanine in position 2 has been replaced with glycineto make the peptide resistant to DPP IV cleavage. The replacement ofalanine is shown to increase the stability and potency of the peptide.The patent application describes how the GLP-2 analogue may be usedagainst diseases associated with inflammation and destruction of theintestinal epithelial mucosa. These include massive small intestineresection, inflammatory bowel disease, chemotherapy induced mucositisand ischemic injury.

WO 02/066511 describes GLP-2 analogues having an extended half life invivo and their use as medicaments in the treatment of gastrointestinaldisorders, such as inflammatory bowel diseases.

WO 01/41779 describes the use of h[Gly2]GLP-2 (SEQ ID NO:54) as apretreatment for inhibiting chemotherapy induced apoptosis and promotingcell survival.

All references cited herein are expressly incorporated by reference intheir entirety.

The use of GLP-2 or analogues of GLP-2 in the treatment of variousdiseases has been proposed by many scientists. However, there is still aneed for improved and stable GLP-2 analogues.

SUMMARY OF THE INVENTION

Broadly, the present invention concerns GLP-2 analogues which compriseone of more substitutions as compared to wild-type GLP-2 and which mayhave the property of an improved biological activity in vivo and/orimproved chemical stability, e.g. as assessed in in vitro stabilityassays. More particularly, preferred GLP-2 analogues of the presentinvention comprise substitutions at one or more of positions 8, 16, 24and/or 28 of the wild-type GLP-2 sequence, optionally in combinationwith further substitutions at position 2 (as mentioned in theintroduction) and one or more of positions 3, 5, 7, 10 and 11, and/or adeletion of one or more of amino acids corresponding to positions 31 to33 of the wild-type GLP-2 sequence and/or the addition of a N-terminalor C-terminal stabilizing peptide sequence. As well as providing GLP-2analogues that may have improved chemical stability and/or biologicalactivity, the present invention also relates to providing compounds thathave preferential intestinal growth promoting activity in the smallintestine compared to the colon and vice versa, in particular byincluding modification at one or more of positions Asp3 and/or Ser 8and/or Asn16 and/or Asn24 and/or Gln28 of wild-type GLP-2.

Accordingly, in one aspect, the present invention provides a GLP-2analogue which is represented by general Formula I (SEQ ID NO:51):R¹-Z¹-His-X2-X3-Gly-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-Ala-X19-X20-X21-Phe-Ile-X24-Trp-Leu-Ile-X28-Thr-Lys-X31-X32-X33-Z²-R²wherein:

-   R¹ is hydrogen, C₁₋₄ alkyl (e.g. methyl), acetyl, formyl, benzoyl or    trifluoroacetyl-   X2 is Gly, Ala or Sar-   X3 is Glu or Asp-   X5 is Ser or Thr-   X6 is Phe or Pro-   X7 is Ser or Thr-   X8 is Asp or Ser-   X9 is Glu or Asp-   X10 is Met, Leu, Nle or an oxidatively stable Met-replacement amino    acid-   X11 is Asn, Ala, Lys or Ser-   X12 is Thr or Lys-   X13 is Ile, Glu or Gln-   X14 is Leu, Met or Nle-   X15 is Asp or Glu-   X16 is Asn or Ala-   X17 is Leu or Glu-   X18 is Ala or Aib-   X19 is Ala or Thr-   X20 is Arg or Lys-   X21 is Asp or Ile-   X24 is Asn, Ala or Glu-   X28 is Gln, Ala or Asn-   X31 is Pro, Ile or deleted-   X32 is Thr or deleted-   X33 is Asp, Asn or deleted-   R² is NH₂ or OH;-   Z¹ and Z² are independently absent or a peptide sequence of 3-20    amino acid units selected from the group consisting of Ala, Leu,    Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met and Orn;    wherein the GLP-2 analogue comprises one or more of substitutions    selected from X8 is Ser and/or X16 is Ala and/or X24 is Ala and/or    X28 is Ala;    or a pharmaceutically acceptable salt or derivative thereof.

In a further embodiment, the present invention provides a GLP-2 analoguerepresented by general Formula II (SEQ ID NO:52):R¹-Z¹-His-Gly-X3-Gly-X5-Phe-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-Ala-X19-Arg-Asp-Phe-Ile-X24-Trp-Leu-Ile-X28-Thr-Lys-X31-X32-X33-Z²-R²wherein:

-   R¹ is hydrogen, C₁₋₄ alkyl (e.g. methyl), acetyl, formyl, benzoyl or    trifluoroacetyl-   X3 is Glu or Asp-   X5 is Ser or Thr-   X7 is Ser or Thr-   X8 is Asp or Ser-   X9 is Glu or Asp-   X10 is Met, Leu, Nle or an oxidatively stable Met-replacement amino    acid-   X11 is Asn, Ala, Lys or Ser-   X12 is Thr or Lys-   X13 is Ile, Glu or Gln-   X14 is Leu, Met or Nle-   X15 is Asp or Glu-   X16 is Asn or Ala-   X17 is Leu or Glu-   X19 is Ala or Thr-   X24 is Asn or Ala-   X28 is Gln, Ala or Asn-   X31 is Pro, Ile or deleted-   X32 is Thr or deleted-   X33 is Asp or deleted-   R² is NH₂ or OH;-   Z¹ and Z² are independently absent or a peptide sequence of 3-20    amino acid units selected from the group consisting of Ala, Leu,    Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met and Orn;    wherein the GLP-2 analogue comprises one or more of substitutions    selected from X8 is Ser and/or X16 is Ala and/or X24 is Ala and/or    X28 is Ala;    or a pharmaceutically acceptable salt or derivative thereof.

In a further embodiment, the present invention provides a GLP-2 analoguerepresented by general Formula III (SEQ ID NO:53):R¹-Z¹-His-Gly-X3-Gly-X5-Phe-X7-X8-Glu-X10-X11-Thr-Ile-Leu-Asp-X16-Leu-Ala-Ala-Arg-Asp-Phe-Ile-X24-Trp-Leu-Ile-X28-Thr-Lys-X31-X32-X33-Z²-R²wherein:

-   R¹ is hydrogen, C₁₋₄ alkyl (e.g. methyl), acetyl, formyl, benzoyl or    trifluoroacetyl-   X3 is Glu or Asp-   X5 is Ser or Thr-   X7 is Ser or Thr-   X8 is Asp or Ser-   X10 is Met, Leu, Nle, or an oxidatively stable Met-replacement amino    acid-   X11 is Asn, Ala, Lys or Ser-   X24 is Asn or Ala-   X28 is Gln or Ala-   X31 is Pro or deleted-   X32 is Thr or deleted-   X33 is Asp or deleted-   R² is NH₂ or OH;-   Z¹ and Z² are independently absent or a peptide sequence of 3-20    amino acid units selected from the group consisting of Ala, Leu,    Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met and Orn    wherein the GLP-2 analogue comprises one or more of substitutions    selected from X8 is Ser and/or X16 is Ala and/or X24 is Ala and/or    X28 is Ala;    or a pharmaceutically acceptable salt or derivative thereof. Where    X16 is not Ala, it will be Asn.

In certain embodiments, when Z¹ is present R¹ may be H, and when Z² ispresent R² may be OH.

In some embodiments of the present invention, the GLP-2 analogue atleast 60% amino acid sequence identity to wild-type GLP-2 (1-33) havingthe sequence set out in the introduction of the application (SEQ IDNO:50), more preferably at least 63% sequence identity, more preferablyat least 66% sequence identity and still more preferably at least 69%sequence identity.

“Percent (%) amino acid sequence identity” with respect to the GLP-2polypeptide sequences is defined as the percentage of amino acidresidues in a candidate sequence that are identical with the amino acidresidues in the wild-type GLP-2 sequence, after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percentsequence identity, and not considering any conservative substitutions aspart of the sequence identity. Sequence alignment can be carried out bythe skilled person using techniques well known in the art for exampleusing publicly available software such as BLAST, BLAST2 or Alignsoftware, see: Altschul et al (Methods in Enzymology, 266:460-480(1996)) or Pearson et al (Genomics, 46, 24,36, 1997) for the Alignprogram. The percentage sequence identities used herein and inaccordance with the present invention are determined using theseprograme with their default settings. More generally, the skilled personcan readily determine appropriate parameters for determining alignment,including any algorithms needed to achieve maximal alignment over thefull length of the sequences being compared.

In some preferred embodiments, the GLP-2 peptide analogues representedby Formula I, II or III comprise substitutions at more than one ofpositions X8, X16, X24 and/or X28 and/or a combination of thesesubstitutions with other substitutions, preferably those at positionsX3, X5, X7, X10 and/or X11.

Examples of combinations of X8, X16, X24 and/or X28 substitutions thatfall within Formulae I to III include:

-   Ser8, Ala16-   Ser8, Ala24-   Ser8, Ala28-   Ala16, Ala24-   Ala16, Ala28-   Ala24, Ala28-   Ser8, Ala16, Ala24-   Ser8, Ala16, Ala28-   Ser8, Ala24, Ala28-   Ala16, Ala24, Ala28-   Ser8, Ala16, Ala24, Ala28

Examples of substitutions at positions X3, X5, X7, X10 and/or X11 thatfall within Formulae I to III and may be combined with a substitution atone or more of positions X8, X16, X24 and/or X28 include:

-   Glu3, Leu10, Ala11,24-   Glu3, Thr5, Leu10, Ser11, Ala16,24,28-   Glu3, Thr5, Leu10, Lys11, Ala16,24,28-   Glu3, Thr5, Ser8, Leu 10, Lys11, Ala16,24,28-   Glu3, Thr5, Ser8,11, Leu10, Ala16,24,28-   Glu3, Thr5, Ser8,11, Leu10, Ala16,24,28-   Glu3, Ser8,11, Leu10, Ala16,24,28-   Glu3, Leu10, Ser 1, Ala16,24,28-   Glu3, Leu10, Lys11, Ala16,24,28-   Glu3, Thr5, Leu10, Ala11,16,24,28-   Glu3, Thr5, Leu10, Ala11,16,24,28, Ile21-   Glu3, Thr5, Ser8, Leu 10, Ala 11,16,24,28-   Glu3, Ser8, Leu10, Ala11,16,24,28-   Glu3, Leu10, Ala11,16,24,28-   Thr7, Leu10, Ala11, 24-   Thr7, Leu10, Lys11, Ala24-   Thr7, Leu10, Ser11, Ala24-   Thr7, Leu10, Ser8,11, Ala24-   Thr7, Ser8, Leu10, Ala11,24-   Thr7, Ser8, Leu10, Lys11, Ala24-   Ser8, Leu10, Ala11,24-   Leu10, Ala24-   Leu10, Ala11, Ala24-   Leu10, Ala11,24,28-   Leu10, Ala11,16,24,28-   Leu10, Lys11, Ala24-   Leu10, Ser11, Ala24-   Leu10, Ser8,11, Ala24    or a deletion at one or more of positions X31-X33 in combination    with an above mentioned change at position 8, 16, 24 and/or 28.

Specific examples of the GLP-2 compounds of the present invention areset out in the detailed description below.

As well as providing GLP-2 analogues that may have improved chemicalstability and biological activity, the present invention also relates toproviding compounds that have preferential growth promoting activity inthe small intestine compared to the colon and vice versa. In particular,the experiments described herein show that substitution at positionsAsp3 and/or Ser 8 and/or Asn16 and/or Gln28 of wild-type GLP-2 provide apreferential increase of the small intestine weight when administered totest animals compared to the increase in colon mass. These findings meanthat the exemplified compounds may be useful for treating conditionswhere it is advantageous to have an increased growth promoting effect inthe small intestine, while having a lesser effect on the colon, and viceversa.

Thus, compounds that are preferred for causing growth of the smallintestine typically comprise one or more substitutions at positions 3,8, 16 and/or 28 of wild-type GLP-2. Such compounds may selectively causegrowth of the small intestine rather than the colon. They may thereforebe used for conditions affecting or related to the small intestine.

Preferably, such small intestine-selective compounds comprisesubstitutions at more than one of positions X3, X7, X16, X24, X28, X31,X32 and/or X33. Thus, the small-intestine-selective compounds maycomprise more than one of the substitutions X3 is Glu, X7 is Ser, X16 isAla, X24 is Ala, X28 is Ala, X31 is Ile, X32 is Thr and X33 is Asp. Theamino acid residues in positions X31, X32 and X33 may optionally bedeleted.

Exemplified compounds preferentially stimulating epithelial growth inthe small intestine include 1809 (SEQ ID NO:1), 1818 (SEQ ID NO:8), 1819(SEQ ID NO:9), 1820 (SEQ ID NO:10), 1826 (SEQ ID NO:16), 1827 (SEQ IDNO:17), 1844 (SEQ ID NO:32), 1845 (SEQ ID NO:33), 1846 (SEQ ID NO:34),1848 (SEQ ID NO:36), 1849 (SEQ ID NO:37), 1850 (SEQ ID NO:38), 1851 (SEQID NO:39), 1852 (SEQ ID NO:40), 1853 (SEQ ID NO:41), 1855 (SEQ IDNO:42), 1857 (SEQ ID NO:45), 1858 (SEQ ID NO:46), 1859 (SEQ ID NO:47)(see Table 1).

On the other hand, compounds of the present invention that do not havethese modifications, e.g. which comprise one or more substitutions atpositions 10, 11 and/or 24, may be preferred for inducing preferentialgrowth of the colon rather than the small intestine. They may thereforebe used for treatment of conditions affecting or related to the colon.

Such colon-selective compounds may comprise more than one of thesubstitutions at positions X3, X8 and/or X24. For example, they maycomprises more than one substitution selected from X3 is Asp, X8 is Aspand X24 is Ala. The amino acid residues in positions X31, X32 and X33may optionally be deleted.

Exemplified compounds preferentially stimulating epithelial growth inthe colon include 1830 (SEQ ID NO:20), 1831 (SEQ ID NO:21), 1835 (SEQ IDNO:25), 1836 (SEQ ID NO:26), 1839 (SEQ NO:27), 1840 (SEQ ID NO:28), 1841(SEQ ID NO:29), and 1843 (SEQ ID NO:31).

Exemplified compounds without preferential growth in small intestine orcolon: [Gly2]GLP-2 (i.e., reference molecule), 1559 (SEQ ID NO:54), 1821(SEQ ID NO:11), 1822 (SEQ ID NO:12), 1823 (SEQ ID NO:13), 1825 (SEQ IDNO:15), 1828 (SEQ ID NO:18), 1829 (SEQ ID NO:19), 1832 (SEQ ID NO:22),1833 (SEQ ID NO:23), 1834 (SEQ ID NO:24), 1842 (SEQ ID NO:30), 1854 (SEQID NO:42).

The compounds of the invention also have increased chemical stability,e.g. against acid hydrolysis, oxidation and deamidation. It is believedthat substitutions at positions X3 and/or X33 may improve stability toacid hydrolysis. Substitution at position X10 may improve oxidativestability. Substitution at one or more of positions X11, X16 and/or X24may increase stability against deamidation. The GLP-2 analogue of theinvention may therefore exhibit enhanced stability towards degradationin acidic solution, towards deamidation, and/or towards oxidativedegradation, relative to Gly2-GLP-2. Preferably, the GLP-2 analoguemaintains an observed purity of at least (40%, 50%, 60%, 70%, 80%, or90%, 95%, or 99% relative to the initial purity in at least one of thedegradation tests described in Example 7 below. Additionally oralternatively, it may maintain an observed purity of at least 60%relative to initial purity in a solution of HCl 0.1 M after 12 days.Additionally or alternatively it may maintain an observed purity of atleast 70% relative to initial purity in a solution of NH4HCO3 0.1 Mafter 6 days.

In a further aspect, the present invention provides a compositioncomprising a GLP-2 analogue as defined herein, or a salt or derivativethereof, in admixture with a carrier. In preferred embodiments, thecomposition is a pharmaceutically acceptable composition and the carrieris a pharmaceutically acceptable carrier. The GLP-2 peptide analogue maybe a pharmaceutically acceptable acid addition salt of the GLP-2analogue.

In a further aspect, the present invention provides a GLP-2 analogue asdefined herein, or a salt thereof, for use in therapy.

In a further aspect, the present invention provides use of a GLP-2analogue, or a salt or derivative thereof for the preparation of amedicament for the treatment and/or prevention of stomach andbowel-related disorders, such as the treatment of neonatals withcompromised intestine function, osteoporosis, and DPP-IV(dipeptidylpeptidase-IV) mediated conditions. By way of example, thestomach and bowel-related disorders include ulcers, gastritis, digestiondisorders, malabsorption syndromes, short-gut syndrome, cul-de-sacsyndrome, inflammatory bowel disease, celiac sprue (for example arisingfrom gluten induced enteropathy or celiac disease), tropical sprue,hypogammaglobulinemic sprue, enteritis, regional enteritis (Crohn'sdisease), ulcerative colitis, irritable bowel syndrome associated withdiarrhea, small intestine damage and short bowel syndrome.

Other conditions that may be treated with the GLP-2 analogues of theinvention, or for which the GLP-2 analogues may be usefulprophylactically or therapeutically, include radiation enteritis,infectious or post-infectious enteritis, and small intestinal damage dueto toxic or other chemotherapeutic agents. This may requireadministration of the GLP-2 analogue prior to, concurrently with orfollowing a course of chemotherapy or radiation therapy in order toreduce side effects of chemotherapy such as diarrhea, abdominal crampingand vomiting, and reduce the consequent structural and functional damageof the intestinal epithelium resulting from the chemotherapy orradiation therapy.

The invention therefore also provides a therapeutic kit comprising acancer chemotherapy drug and a GLP-2 analogue of the present invention,each optionally in combination with a pharmaceutically acceptablecarrier. The two therapeutic agents may be packaged separately (e.g. inseparate vials) for separate administration, or may be provided in thesame composition. Thus the invention further provides a pharmaceuticalcomposition comprising a cancer chemotherapy drug and a GLP-2 analogueof the present invention in combination with a pharmaceuticallyacceptable carrier.

For patients having gastrointestinal mucosal neoplasia, or an increasedrisk of gastrointestinal mucosal neoplasia, it may be desirable toselect a compound so as to reduce or abrogate the risk of reduced sideeffects such as stimulation or aggravation of gastrointestinal mucosalneoplasia. For example, when selecting a compound for treating a patientwith colon neoplasia (whether benign or malignant), or at risk ofdeveloping colon neoplasia, it may be more appropriate to select acompound which is selective for the small intestine over the colon thana non-selective compound or a compound which is selective for the colonover the small intestine.

In other aspects, the present invention provides the use of the GLP-2analogues for the preparation of a medicament for the treatment and/orprevention of malnutrition, for example conditions such as the wastingsyndrome cachexia and anorexia.

In a further aspect, the present invention provides a nucleic acidmolecule comprising a nucleic acid sequence encoding a GLP-2 analogue ofas defined herein.

In further aspects, the present invention provides an expression vectorcomprising the above nucleic acid sequence, optionally in combinationwith sequences to direct its expression, and host cells transformed withthe expression vectors. Preferably the host cells are capable ofexpressing and secreting the GLP-2 analogue. In a still further aspect,the present invention provides a method of producing the GLP-2 analogue,the method comprising culturing the host cells under conditions suitablefor expressing the GLP-2 analogue and purifying the GLP-2 analogue thusproduced.

The invention further provides a nucleic acid of the invention, anexpression vector of the invention, or a host cell capable of expressingand secreting a GLP-2 analogue of the invention, for use in therapy. Itwill be understood that the nucleic acid, expression vector and hostcells may be used for treatment of any of the disorders described hereinwhich may be treated with the GLP-2 analogues themselves. References toa therapeutic composition comprising a GLP-2 analogue of the invention,or administration of a GLP-2 analogue of the invention, should thereforebe construed to encompass administration of a nucleic acid, expressionvector or host cell of the invention except where the context demandsotherwise.

In a further aspect, the present invention provides a method of treatinga stomach and bowel-related disorder in a patient in need thereof byadministering an effective amount a GLP-2 analogue as defined herein, ora salt or derivative thereof, or a nucleic acid, expression vector orhost cell of the invention. Examples of stomach and bowel-relateddisorders are provided above.

In a further aspect, the present invention provides a method of treatingor preventing a side effect of chemotherapy or radiation therapy in apatient in need thereof, the method comprising administering aneffective amount a GLP-2 analogue as defined herein, or a salt orderivative thereof, or a nucleic acid, expression vector or host cell ofthe invention.

In a further aspect, the present invention provides a method of treatingor preventing malnutrition, for example conditions such as the wastingsyndrome cachexia and anorexia, in a patient in need thereof, the methodcomprising administering an effective amount a GLP-2 analogue as definedherein, or a salt or derivative thereof, or a nucleic acid, expressionvector or host cell of the invention.

Embodiments of the present invention will now be described in moredetail by way of examples and not limitation with reference to theaccompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a set of graphs showing full dose-response data of four smallintestine selective compounds (compound nos. 1846 (SEQ ID NO:34), 1855(SEQ ID NO:43), 1848 (SEQ ID NO:36), and 1858 (SEQ ID NO:46) on smallintestinal (SI) mass in C57BL mice. Compounds were administered bysubcutaneous injection b.i.d. for three days at the following doses: 0(vehicle), 5, 15, 45, 135, 405 nmol/kg (n=6/dose group). Responses ateach dose level were compared with responses obtained at the same doselevel in pair-treated mice treated with the non-selective referencecompound [Gly2]GLP-2 (SEQ ID NO:54). To correct for changes in bodyweight (BW), SI mass was expressed relative to BW (SI-BW ratio) and thegrowth response at each dose level was normalized to the responseobserved with the non-selective reference compound [Gly2]GLP-2 (SEQ IDNO:54). Results demonstrated that within the dose range 5-405 nmol/kg,the dose-response relationships of the small intestine selectivecompounds 1846 (SEQ ID NO:34), 1855 (SEQ ID NO:43), 1848 (SEQ ID NO:36),and 1858 (SEQ ID NO:46) were significantly different from thenon-selective reference compound [Gly2]GLP-2 (SEQ ID NO:54) (p<0.05 intwo-way ANOVA) and all four compounds stimulated small intestinal growthwith maximal responses that were significant greater than [Gly2]GLP-2(SEQ ID NO:54). Values are means±SEM. *:P<0.05 relative to the equimolardose of [Gly2]GLP-2 (SEQ ID NO:54).

FIG. 2 is a set of graphs showing full dose-response data of four smallintestine selective compounds (compound nos. 1846 (SEQ ID NO:34), 1855(SEQ ID NO:43), 1848 (SEQ ID NO:36), 1858 (SEQ ID NO:46)) on the SmallIntestine (SI)-to-Colon Sensitivity Index mice relative to thenon-selective reference compound [Gly2]GLP-2 (SEQ ID NO:54). Compoundswere administered by subcutaneous injection b.i.d. for three days inC57BL mice at the following doses: 0 (vehicle), 5, 15, 45, 135, 405nmol/kg (n=6/dose group). Responses at each dose level were comparedwith responses obtained at the same dose level in pair-treated micetreated with the non-selective reference compound [Gly2]GLP-2 (SEQ IDNO:54). The SI-Colon Sensitivity Index was calculated as SI-massrelative to colon mass and the Sensitivity Index at each dose level wasnormalized to the response observed with the non-selective referencecompound [Gly2]GLP-2 (SEQ ID NO:54). Results demonstrated that withinthe dose range 5-405 nmol/kg, the dose-response relationships of thesmall intestine selective compounds 1857 (SEQ ID NO:45) and 1820 (SEQ IDNO:10) were significantly different from the non-selective referencecompound [Gly2]GLP-2 (SEQ ID NO:54) (p<0.05 in two-way ANOVA) andcompounds 1846 (SEQ ID NO:34), 1855 (SEQ ID NO:43), and 1848 (SEQ IDNO:36) demonstrated increased maximal small intestinal sensitivityrelative to [Gly2]GLP-2. Values are means±SEM. *:P<0.05 relative to theequimolar dose of [Gly2]GLP-2.

FIG. 3 is a set of graphs showing full dose-response data of two smallintestine selective compounds (compound nos. 1857 (SEQ ID NO:45), 1849(SEQ ID NO:37), and 1820 (SEQ ID NO:10) on small intestinal (SI) mass inC57BL mice. Compounds were administered by subcutaneous injection b.i.d.for three days at the following doses: 0 (vehicle), 5, 15, 45, 135, 405nmol/kg (n=6/dose group). Responses at each dose level were comparedwith responses obtained at the same dose level in pair-treated micetreated with the non-selective reference compound [Gly2]GLP-2. Tocorrect for changes in body weight (BW), SI mass was expressed relativeto BW (SI-BW ratio) and the growth response at each dose level wasnormalized to the response observed with the non-selective referencecompound [Gly2]GLP-2 (SEQ ID NO:54). Results demonstrated that withinthe dose range 5-405 nmol/kg, the dose-response relationships of thesmall intestine selective compounds 1857 (SEQ ID NO:45), 1849 (SEQ IDNO:37), and 1820 (SEQ ID NO:10) were significantly different from thenon-selective reference compound [Gly2]GLP-2 (SEQ ID NO:54) (p<0.05 intwo-way ANOVA) and both compounds stimulated small intestinal growthwith maximal responses that were significant greater than [Gly2]GLP-2(SEQ ID NO:54). Values are means±SEM. *:P<0.05 relative to the equimolardose of [Gly2]GLP-2 (SEQ ID NO:54).

FIG. 4 is a set of graphs showing full dose-response data of three smallintestine selective compounds (compound nos. 1857 (SEQ ID NO:45), 1820(SEQ ID NO:10), and 1849 (SEQ ID NO:37)) on the Small Intestine(SI)-to-Colon Sensitivity Index mice relative to the non-selectivereference compound [Gly2]GLP-2 (SEQ ID NO:54). Compounds wereadministered by subcutaneous injection b.i.d. for three days in C57BLmice at the following doses: 0 (vehicle), 5, 15, 45, 135, 405 nmol/kg(n=6/dose group). Responses at each dose level were compared withresponses obtained at the same dose level in pair-treated mice treatedwith the non-selective reference compound [Gly2]GLP-2 (SEQ ID NO:54).The SI-Colon Sensitivity Index was calculated as SI-mass relative tocolon mass and the Sensitivity Index at each dose level was normalizedto the response observed with the non-selective reference compound[Gly2]GLP-2 (SEQ ID NO:54).

Results demonstrated that within the dose range 5-405 nmol/kg, thedose-response relationships of the small intestine selective compounds1857 (SEQ ID NO:45), 1820 (SEQ ID NO:10), and 1849 (SEQ ID NO:37) weresignificantly different from the non-selective reference compound[Gly2]GLP-2 (SEQ ID NO:54) (p<0.05 in two-way ANOVA) and all threecompounds demonstrated increased maximal small intestinal sensitivityrelative to [Gly2]GLP-2 (SEQ ID NO:54). Values are means±SEM. *:P<0.05relative to the equimolar dose of [Gly2]GLP-2 (SEQ ID NO:54).

FIG. 5 shows examples of full dose-response data of a non-selectivecompound ([Gly2]GLP-2 (SEQ ID NO:54)), and a small intestine selectivecompound (Compound 1848 (SEQ ID NO:36)), on small intestine, colon andstomach mass. Compounds were administered by subcutaneous injectionb.i.d. for three days in C57BL mice at the following doses: 0 (vehicle),5, 15, 45, 135, 405 nmol/kg (n=6/dose group). Results demonstrated thatwithin the dose range 5-405 nmol/kg, [Gly2]GLP-2 (SEQ ID NO:54) produceddose-dependent growth stimulation in small intestine, colon and stomach,while Compound 1848 (SEQ ID NO:36) only produced dose-dependent growthstimulation in the small intestine. Values are means±SEM. *:P<0.05relative to the vehicle.

FIG. 6 is a set of graphs showing the effect of compound 1846 (SEQ IDNO:34) (5, 15, 45, 135, 405 and 810 nmol/kg, s.c., b.i.d.; n=6/dosegroup) on A) small intestine-to-body weight ratio (SI-BW), and B) smallintestinal length (SI length) in mice treated with the cytostatic drug5-Fluorouracil (5-FU). Compound 1846 (SEQ ID NO:34) was administered for3 days prior to and for 4 days together with 5-FU, 50 mg/kg, i.p. daily.For reference, [Gly2]GLP-2 (SEQ ID NO:54) (405 nmol/kg) was given to onegroup of animals. Results from control animals treated with vehicle(PBS) or 5-FU alone are also shown. Compound 1846 (SEQ ID NO:34)prevented 5-FU-induced small intestine atrophy (reduced organ mass andshortening) in C57BL mice. Values are means±SEM. *P<0.05, relative to5-FU.

FIG. 7 is a set of graphs showing the effect of compound 1848 (SEQ IDNO:36) (5, 15, 45, 135, 405 and 810 nmol/kg, s.c., b.i.d.; n=6/dosegroup) on A) small intestine-to-body weight ratio (SI-BW), and B) smallintestinal length (SI length) in mice treated with the cytostatic drug5-Fluorouracil (5-FU). Compound 1848 (SEQ ID NO:36) was administered for3 days prior to and for 4 days together with 5-FU, 50 mg/kg, i.p. daily.For reference, [Gly2]GLP-2 (SEQ ID NO:54) (405 nmol/kg) was given to onegroup of animals. Results from control animals treated with vehicle(PBS) or 5-FU alone are also shown. Compound 1848 (SEQ ID NO:36)prevented 5-FU-induced small intestine atrophy (reduced organ mass andshortening) in C57BL mice and high doses of compound 1848 (SEQ ID NO:36)were more efficacious than [Gly2]GLP-2. Values are means±SEM. *P<0.05,relative to 5-FU. #P<0.05, relative to [Gly2]GLP-2.

FIG. 8 is a set of graphs showing the effect of compound 1855 (SEQ IDNO:43) (5, 15, 45, 135, and 405 nmol/kg, s.c., b.i.d.; n=6/dose group)on A) small intestine-to-body weight ratio (SI-BW), and B) smallintestinal length (SI length) in mice treated with the cytostatic drug5-Fluorouracil (5-FU). Compound 1855 (SEQ ID NO:43) was administered for3 days prior to and for 4 days together with 5-FU, 50 mg/kg, i.p. daily.For reference, [Gly2]GLP-2 (405 nmol/kg) was given to one group ofanimals. Results from control animals treated with vehicle (PBS) or 5-FUalone are also shown. Compound 1855 (SEQ ID NO:43) prevented5-FU-induced small intestine atrophy (reduced organ mass and shortening)in C57BL mice and the highest dose of compound 1855 (SEQ ID NO:43) wasmore efficacious than the equimolar dose of [Gly2]GLP-2 (SEQ ID NO:54).Values are means±SEM. *P<0.05, relative to 5-FU. #P<0.05, relative to[Gly2]GLP-2 (SEQ ID NO:54).

FIG. 9 is a set of graphs showing the effect of compound 1857 (SEQ IDNO:45) (5, 15, 45, 135, and 405 nmol/kg, s.c., b.i.d.; n=6/dose group)on A) small intestine-to-body weight ratio (SI-BW), and B) smallintestinal length (SI length) in mice treated with the cytostatic drug5-Fluorouracil (5-FU). Compound 1857 (SEQ ID NO:45) was administered for3 days prior to and for 4 days together with 5-FU, 50 mg/kg, i.p. daily.For reference, [Gly2]GLP-2 (SEQ ID NO:54) (405 nmol/kg) was given to onegroup of animals. Results from control animals treated with vehicle(PBS) or 5-FU alone are also shown. Compound 1857 (SEQ ID NO:45)prevented 5-FU-induced small intestine atrophy (reduced organ mass andshortening) in C57BL mice and the highest doses of compound 1855 (SEQ IDNO:43) were more efficacious than a high dose of [Gly2]GLP-2 (SEQ IDNO:54). Values are means±SEM. *P<0.05, relative to 5-FU. #P<0.05,relative to [Gly2]GLP-2 (SEQ ID NO:54).

FIG. 10 is a set of graphs showing effects of compound 1846 (SEQ IDNO:34) (16, 80 and 400 nmol/kg/d; s.c., n=6/dose group) in maleSpague-Dawley rats treated with the cytostatic drug 5-Fluorouracil(5-FU). Compound 1846 (SEQ ID NO:34) was administered for 3 days priorto and for 4 days together with 5-FU (75 mg/kg, i.p. daily). Resultswere compared with responses in control animals treated with vehicle(saline) or 5-FU alone. Saline and 5-FU controls are also shown.Compound 1846 (SEQ ID NO:34) prevented 5-FU-induced small intestineatrophy (reduced small intestinal mass and intestinal shortening) inrats. Values are means±SEM. *P<0.05, relative to 5-FU alone.

FIG. 11 is a set of graphs showing the therapeutic effect of compound1846 (SEQ ID NO:34) on diarrhea induced by treatment with the cytostaticdrug 5-fluorouracil (5-FU). Rats were treated with 5-FU for 4 days with75 mg/kg once daily (days 1-4); (n=40 rats). Half of the animalsreceived additional treatment with compound 1846 (SEQ ID NO:34) (400nmol/kg s.c. once daily) during the last 3 days prior to (days −3 to −1)and during 4 days of 5-FU treatment (days 0 to 3). Rats were observedtwice daily (morning and evening) to determine whether the animal haddiarrhea and the severity of the diarrhea was scored according to thisscale: (0) no diarrhea; (1) mild—fecal staining around the anus; (2)moderate—fecal staining on the hind limbs and tail; and (3) severe—fecalstaining on the front limbs and abdomen. On day 5, about 70% of SpragueDawley rats that received 5-FU alone had developed diarrhea (FIG. 11A)while only 30% of the rats that were co-treated with compound 1846 (SEQID NO:34) developed diarrhea (FIG. 11B). These results indicate thatcompound 1846 (SEQ ID NO:34) effectively prevents injury of the smallintestine and thus the development of diarrhea during cytostatictreatment with 5-FU.

FIG. 12 is a set of graphs showing the effect of compound 1846 (SEQ IDNO:34) on epithelial crypt-villus height (FIG. 12A) and muscularthickness (FIG. 12B) in the jejunum (i.e., mid section of the smallintestine). Sprague Dawley rats were treated intravenously with compound1846 (SEQ ID NO:34) (0.62, 3.41 or 6.2 mg/kg once daily) for 5 days(n=6/dose group). Then the animals were sacrificed and small intestinalbiopsies (1 cm) were collected 25 cm distal from the pyloric sphincter.The biopsies were fixed, embedded in paraffin, sectioned and stainedwith haematoxylin and eosin. Stained sections were examined under amicroscope and crypt depth, villus height, crypt-villus length andmuscularis thickness measured. As illustrated compound 1846 (SEQ IDNO:34) produced dose-dependent increases in crypt-villus length inintestinal epithelium from the jejunum, but had no effect on themuscular thickness of the jejunum. These results suggest that compound1846 (SEQ ID NO:34) primarily exerts its proliferative actions in thesmall intestine through stimulation of small epithelial growth. Valuesare means±SEM. *P<0.05, relative to vehicle controls (0 mg/kg/d).

FIG. 13 is a graph illustrating the effect of compound 1848 (SEQ IDNO:36) on small intestinal ulcers induced by indomethacin. Male SpragueDawley rats were treated with vehicle (saline; group 1) or indomethacin(7 mg/kg s.c., once daily for two days; groups 2-7). Indomethacin wasgiven alone (group 2) or in combination with prednisolone (10 mg/kgs.c.; group 3) or with 8, 40, or 200 nmol/kg compound 1848 (SEQ IDNO:36) s.c. once daily (groups 4-6). Finally, one group of rats wastreated with combination treatment of prednisolone (10 mg/kg s.c.) andcompound 1848 (SEQ ID NO:36), 200 nmol/kg s.c. once daily (group 7).Treatment with compound 1848 (SEQ ID NO:36) was initiated 4 days priorto indomethacin and continued during the two days of indomethacintreatment. On day 3, rats were sacrificed and upon necropsy, the smallintestine was gently flushed with 10% formalin and filled with formalinfor a period of 5 minutes, after which the intestine was cut open alongthe antimesenteric margin and suspended on a polypropylene plate. Anyremaining intestinal contents were carefully removed with a pair oftweezers. After fixation for another 24 hours at room temperature, thetissue was rinsed in Milli-Q water and surface-stained for 20 minuteswith Alcian Green 3BX (Chroma) prepared as a 0.5% solution in 1% aceticacid (Bie & Berntsen). After removal of excess staining solution withMilli-Q water the tissue preparation was transferred to 70% alcohol andanalyzed using a stereomicroscope at low magnification (×7). Starting atthe pylorus, the small intestine was scanned and the shape (circular vs.linear) and size (circular ulcers: diameter, linear ulcers:length×width) of all ulcers was measured using a standard ruler(resolution: 0.5 mm). An ulcer was defined as an area, which lackedepithelial surface. In healing ulcers only the area that still lackedepithelial surface was regarded an ulcer even if the villus structurewas still missing in a larger area. All analysis was performed in ablinded manner. As illustrated in FIG. 13, indomethacin caused a stronginduction of small intestinal ulcers (total small intestinalulceration=333±21 mm²). Treatment with prednisolone caused a significantreduction in the extent of ulceration by app. 29%. Treatment withcompound 1848 (SEQ ID NO:36) prevented indomethacin-induced ulcerationin a dose-dependent fashion and at the highest dose the total ulcerationwas reduced by almost 50% (178±17 mm²). This maximal response tocompound 1846 (SEQ ID NO:34) was greater than the effect of prednisoloneand addition of prednisolone in combination with high dose compound 1848(SEQ ID NO:36) did not produced any additional effect. These resultsindicate that compound 1848 (SEQ ID NO:36) effectively preventsindomethacin-induced ulceration in the small intestine. Values aremeans±SEM. *P<0.05 vs. indomethacin (group 2). #P<0.05 vs. prednisolone(group 3).

FIG. 14 is a set of graphs showing the effect of compound 1848 (SEQ IDNO:36) on small intestinal content of TNF-alpha in rats withindomethacin-induced inflammation in the small intestine. Male SpragueDawley rats were treated with vehicle (saline; group 1) or indomethacin(7 mg/kg s.c., once daily for two days; groups 2-7). Indomethacin wasgiven alone (group 2) or in combination with prednisolone (10 mg/kgs.c.; group 3) or with 8, 40, or 200 nmol/kg compound 1848 (SEQ IDNO:36) s.c. once daily (groups 4-6). Finally, one group of rats wastreated with combination treatment of prednisolone (10 mg/kg s.c.) andcompound 1848 (SEQ ID NO:36), 200 nmol/kg s.c. once daily (group 7).Treatment with compound 1848 (SEQ ID NO:36) was initiated 4 days priorto indomethacin and continued during the two days of indomethacintreatment. On day 3, rats were sacrificed and upon necropsy, the smallintestine Upon necropsy the small intestine was divided into threesegments of equal length corresponding to 1) duodenum and proximaljejunum, 2) mid and distal jejunum and 3) ileum. Samples wereimmediately snap-frozen in liquid N₂ and stored at −80° C. untilanalysis. Homogenization and extraction of cellular protein from smallintestinal segments was performed according to the following procedure:Tissue segments were weighed and added a volume of 1.5 ml per gramtissue ice-cold (4° C.) extraction buffer (10 mM Tris-HCl (Sigma) and 1mM EDTA (J.T. Baker), (pH 7.6) with 0.05% sodium azide (Fluka), 1%Tween-80 (Fluka), 2 mM phenylmethylsulfonyl fluoride (PMSF, Fluka) and 1μg/ml of each of the protease inhibitors aprotinin, leupeptin andpepstatin A (Roche Diagnostics)). The tissue was cut into small bitswith a pair of scissors and homogenized three times for 20 seconds (IKAUltraTurrax T25 homogenizer, Janke & Kunkel) at 9500 rpm withintermittent cooling on ice for 30 seconds. The homogenate wascentrifuged at 20.000×g for 30 minutes at 4° C., the supernatant wascollected and stored at −20° C. until analysis for TNF-α smallintestinal protein extractions were analyzed for TNF-α using acommercially available ELISA kit according to the manufacturer'sinstructions (Rat Tumor Necrosis Factor-α UltraSensitive ELISA kit,Biosource International Inc.). The assay has a detection limit of 0.7pg/ml. Protein extractions were analyzed for total protein content usinga commercially available assay (DC protein assay, Bio-Rad LaboratoriesLtd.). Small intestinal tissue levels of TNF-α were expressed relativeto total protein. Compound 1848 (SEQ ID NO:36) significantly decreasedsmall intestinal tissue levels of the proinflammatory cytokine TNF-α andthis effect was most marked in the proximal segment (first ⅓^(rd) ofsmall intestine). Compound 1848 (SEQ ID NO:36) was more efficacious thanprednisolone in the proximal and mid segments (second 1/31^(rd) of smallintestine). Interestingly, compound 1848 (SEQ ID NO:36) suppressedinflammation in the small intestine more effectively than prednisone,and prednisone had no additive effect when given in combination withcompound 1848 (SEQ ID NO:36). These results suggest that compound 1848(SEQ ID NO:36) has a marked anti-inflammatory potential on diseasesprocesses that affect the small intestine. Values are means±SEM. *P<0.05vs. indomethacin (group 2).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless specified otherwise, the following definitions are provided forspecific terms, which are used in the above written description.

Throughout the description and claims the conventional one-letter andthree-letter codes for natural amino acids are used as well as generallyaccepted three letter codes for other α-amino acids, such as sarcosin(Sar), norleucine (Nle) and α-aminoisobutyric acid (Aib). All amino acidresidues in peptides of the invention are preferably of theL-configuration, However, D-configuration amino acids may also bepresent.

Preferred compounds of the present invention have at least one GLP-2biological activity, in particular in causing growth of the intestine.This can be assessed in in vivo assays, for example as described in theexamples, in which the mass of the intestine, or a portion thereof isdetermined after a test animal has been treated or exposed to a GLP-2analogue.

The GLP-2 analogues of the present invention have one or more amino acidsubstitutions, deletions, inversions, or additions compared with nativeGLP-2 and as defined above. This definition also includes the synonymterms GLP-2 mimetics and/or GLP-2 agonists. Further, the analogue of thepresent invention may additionally have chemical modification of one ormore of its amino acid side groups, α-carbon atoms, terminal aminogroup, or terminal carboxylic acid group. A chemical modificationincludes, but is not limited to, adding chemical moieties, creating newbonds, and removing chemical moieties. Modifications at amino acid sidegroups include, without limitation, acylation of lysine 1-amino groups,N-alkylation of arginine, histidine, or lysine, alkylation of glutamicor aspartic carboxylic acid groups, and deamidation of glutamine orasparagine. Modifications of the terminal amino include, withoutlimitation, the des-amino, N-lower alkyl, N-di-lower alkyl, and N-acylmodifications. Modifications of the terminal carboxy group include,without limitation, the amide, lower alkyl amide, dialkyl amide, andlower alkyl ester modifications. Preferably herein lower alkyl is C₁-C₄alkyl. Furthermore, one or more side groups, or terminal groups, may beprotected by protective groups known to the ordinarily-skilled peptidechemist. The α-carbon of an amino acid may be mono- or di-methylated.

Where they are present, oxidatively stable Met-replacement amino acidmeans one which is selected among the group consisting of Met(O)(methionine sulfoxide), Met(O)₂ (methionine sulfone), Val, Ile, Asn, Glx(Glu or Gln), Tyr, Phe, Trp and preferably Leu, Nle, Ala, Ser, and Gly.

By a peptide with “enhanced stability” is meant a peptide (e.g., a GLP-2peptide described herein) having a increased resistance to degradation(e.g., at least 5%, 10%, 25%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%) lessas compared a reference peptide (e.g., Gly2-GLP-2 (SEQ ID NO:54)) undera given set of conditions over a given time period (e.g., at least 1, 6,or 12, or 1, 2, 3, 5, 6, 8, 10, 12, 15, 20, 30, 45, or 60 days).Alternatively or additionally, stability may be assayed by comparing thepercent purity in a sample subjected to a set of conditions a comparedto the same sample not subjected to the set of conditions. Anyconditions described herein or known in the art may be used to testenhanced stability. In particular embodiments, stability is tested underacidic conditions (e.g., at least 0.01, 0.1, 0.2, 0.4, 0.5, 1.0, 2.0, or5.0 M HCl), basic conditions (e.g., at least 0.01, 0.1, 0.2, 0.4, 0.5M1.0, 2.0, or 5.0 M NaOH), oxidative stress (e.g., at least 0.01% 0.05%,0.1%, 0.2%, 0.5%, 1.0%, 1.5%, 2.0%, 5%, or 10%, 20%, or 30% H₂O₂), ordeamindation conditions (e.g., at least 0.01, 0.1, 0.2, 0.4, 0.5, 1.0,2.0, or 5.0 M NH₄HCO₃). Conditions also include (either alone orincombination with the conditions listed increased temperature (e.g., at30, 35, 37, 40, 45, 50, 60, 70, 80, 100, 150° C.). Exemplary conditionsare described in Table 9 below.

It should be understood that the peptides of the invention might also beprovided in the form of a salt or other derivative. Salts includepharmaceutically acceptable salts such as acid addition salts and basicsalts. Examples of acid addition salts include hydrochloride salts,citrate salts and acetate salts. Examples of basic salts include saltswhere the cation is selected from alkali metals, such as sodium andpotassium, alkaline earth metals, such as calcium, and ammonium ions⁺N(R³)₃(R⁴), where R³ and R⁴ independently designates optionallysubstituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, optionallysubstituted aryl, or optionally substituted heteroaryl. Other examplesof pharmaceutically acceptable salts are described in “Remington'sPharmaceutical Sciences”, 17th edition. Ed. Alfonso R. Gennaro (Ed.),Mark Publishing Company, Easton, Pa., U.S.A., 1985 and more recenteditions, and in the Encyclopaedia of Pharmaceutical Technology.

Other derivatives of the GLP-2 analogues of the invention includecoordination complexes with metal ions such as Mn²⁺ and Zn ²⁺, esterssuch as in vivo hydrolysable esters, free acids or bases, hydrates,prodrugs or lipids. Esters can be formed between hydroxyl or carboxylicacid groups present in the compound and an appropriate carboxylic acidor alcohol reaction partner, using techniques well known in the art.Derivatives which as prodrugs of the compounds are convertible in vivoor in vitro into one of the parent compounds. Typically, at least one ofthe biological activities of compound will be reduced in the prodrugform of the compound, and can be activated by conversion of the prodrugto release the compound or a metabolite of it. Examples of prodrugsinclude the use of protecting groups which may be removed in situreleasing active compound or serve to inhibit clearance of the drug invivo.

When present, Z¹ and Z² each independently represent a peptide sequenceof 3-20 or 4-20 amino acid residues, e.g. in the range of 4-15, morepreferably in the range of 4-10 in particular in the range of 4-7 aminoacid residues, e.g., of 4, 5, 6 or 7 amino acid residues, such as 6amino acid residues. Each of the amino acid residues in the peptidesequences Z may independently be selected from Ala, Leu, Ser, Thr, Tyr,Asn, Gln, Asp, Glu, Lys, Arg, His, Met, Orn. Preferably, the amino acidresidues are selected from Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg,His, Orn, and Met, as well as amino acids falling within formula I asdefined in WO01/04156, e.g., Dbu (2,4 diaminobutyric acid) or Dpr(2,3-diaminopropanoic acid), more preferably from Glu, Lys, and Met,especially Lys. The above-mentioned amino acids may have either D- orL-configuration, but preferably the above-mentioned amino acids have anL-configuration. Particularly preferred sequences Z are sequences offour, five or six consecutive lysine residues, and particularly sixconsecutive lysine residues. Exemplary sequences Z are shown in WO01/04156.

In certain embodiments, Z¹ is absent. In such cases, Z² may be eitherpresent or absent.

The present invention includes the following peptides further describedin the experimental section below.

Reference GLP-2 analogue 1559 H-[Gly2]hGLP-2-OHH-HGDGSFSDEMNTILDNLAARDFINWLIQTKITD- (SEQ ID NO:54) OH Examples of GLP-2analogues of the present invention 1809 [Gly2, Glu3, Thr5, Leu10, Ala11,16, 24, 28]hGLP-2-(Lys)₆-NH₂ HGEGTFSDELATILDALAARDFIAWLIATKITDK₆- (SEQID NO:1) NH₂ 1810 [Gly2, Glu3, Thr5, Leu10, Ala11, 16, 24, 28,Ile21]hGLP-2 (1-30)-(Lys)₆-NH₂ HGEGTFSDELATILDALAARIFIAWLIATK₆-NH₂ (SEQID NO:2) 1811 [Gly2, Pro6, Leu10, Ala11, 16, 24, 28]hGLP-2-NH₂HGDGSPSDELATILDALAARDFIAWLIATKITD- (SEQ ID NO:3) NH₂ 1812 [Gly2, Glu3,Leu10, Ala11, 24] hGLP-2-NH₂ HGEGSFSDELATILDNLAARDFIAWLIQTKITD- (SEQ IDNO:4) NH₂ 1813 [Gly2, Leu10, Ala11, 16, 24, 28]hGLP-2-NH₂H-HGDGSFSDELATILDALAARDFIAWLIATKITD- (SEQ ID NO:5) NH₂ 1814 [Gly2,Leu10, Ala11, 24, 28] hGLP-2-NH₂ H-HGDGSFSDELATILDNLAARDFIAWLIATKITD-(SEQ ID NO:6) NH₂ 1815 [Gly2, Glu3, Leu10, Ala11, 16, 24, 28]hGLP-2-NH₂H-HGEGSFSDELATILDALAARDFIAWLIATKITD- (SEQ ID NO:7) NH₂ 1818 [Gly2, Ser8,Leu10, Ala11, 24] hGLP-2(1-30)-K₆-NH₂ H-HGDGSFSSELATILDNLAARDFIAWLIQTK₆-(SEQ ID NO:8) NH₂ 1819 [Gly2, Leu10, Ser11, Ala24] hGLP-2(1-30)-K₆-NH₂H-HGDGSFSDELSTILDNLAARDFIAWLIQTK₆- (SEQ ID NO:9) NH₂ 1820 [Gly2, Thr7,Ser8, Leu10, Ala11, 24]hGLP-2(1-30)-K₆-NH₂H-HGDGSFTSELATILDNLAARDFIAWLIQTK₆- (SEQ ID NO:10) NH₂ 1821 [Gly2, Leu10,Lys11, Ala24] hGLP-2(1-30)-K₆-NH₂ H-HGDGSFSDELKTILDNLAARDFIAWLIQTK₆-(SEQ ID NO:11) NH₂ 1822 [Gly2, Thr7, Leu10, Lys11,Ala24]hGLP-2(1-30)-K₆-NH₂ H-HGDGSFTDELKTILDNLAARDFIAWLIQTK₆- (SEQ IDNO:12) NH₂ 1823 [Gly2, Thr7, Ser8, Leu10, Lys11,Ala24]hGLP-2(1-30)-K₆-NH₂ H-HGDGSFTSELKTILDNLAARDFIAWLIQTK₆- (SEQ IDNO:13) NH₂ 1824 [Gly2, Thr7, Leu10, Ala11, 24] hGLP-2(1-30)-K₆-NH₂H-HGDGSFTDELATILDNLAARDFIAWLIQTK₆- (SEQ ID NO:14) NH₂ 1825 [Gly2, Ser8,Leu10, Ala11, 24]hGLP-2(1-30)-NH₂ H-HGDGSFSSELATILDNLAARDFIAWLIQTK-NH₂(SEQ ID NO:15) 1826 [Gly2, Leu10, Ala24]hGLP-2-K₆- NH₂H-HGDGSFSDELNTILDNLAARDFIAWLIQTKITD (SEQ ID NO:16) K₆-NH₂ 1827 [Gly2,Thr7, Leu10, Ser11, Ala24]hGLP-2(1-30)-K₆-NH₂H-HGDGSFTDELSTILDNLAARDFIAWLIQTK₆- (SEQ ID NO:17) NH₂ 1828 [Gly2, Thr7,Leu10, Ser8, 11, Ala24]hGLP-2(1-30)-K₆-NH₂H-HGDGSFTSELSTILDNLAARDFIAWLIQTK₆- (SEQ ID NO:18) NH₂ 1829 [Gly2, Leu10,Ser8, 11, Ala24] hGLP-2(1-30)-K₆-NH₂ H-HGDGSFSSELSTILDNLAARDFIAWLIQTK₆-(SEQ ID NO:19) NH₂ 1830 [Gly2, Leu10, Ser11, Ala24] hGLP-2(1-30)-NH₂H-HGDGSFSDELSTILDNLAARDFIAWLIQTK-NH₂ (SEQ ID NO:20) 1831 [Gly2, Thr7,Leu10, Ser11, Ala24]hGLP-2(1-30)-NH₂H-HGDGSFTDELSTILDNLAARDFIAWLIQTK-NH₂ (SEQ ID NO:21) 1832 [Gly2, Thr7,Leu10, Ser8, 11, Ala24]hGLP-2(1-30)-NH₂H-HGDGSFTSELSTILDNLAARDFIAWLIQTK-NH₂ (SEQ ID NO:22) 1833 [Gly2, Leu10,Ser8, 11, Ala24] hGLP-2(1-30)-NH₂ H-HGDGSFSSELSTILDNLAARDFIAWLIQTK-NH₂(SEQ ID NO:23) 1834 [Gly2, Thr7, Ser8, Leu10, Ala11, 24]hGLP-2(1-30)-NH₂H-HGDGSFTSELATILDNLAARDFIAWLIQTK-NH₂ (SEQ ID NO:24) 1835 [Gly2, Leu10,Lys11, Ala24] hGLP-2(1-30)-NH₂ H-HGDGSFSDELKTILDNLAARDFIAWLIQTK-NH₂ (SEQID NO:25) 1836 [Gly2, Thr7, Leu10, Lys11, Ala24]hGLP-2(1-30)-NH₂H-HGDGSFTDELKTILDNLAARDFIAWLIQTK-NH₂ (SEQ ID NO:26) 1839 [Leu10, Ala11,Ala24]hGLP-2 (1-33)-K₆-NH₂ H-HGDGSFSDELATILDNLAARDFIAWLIQTKITD (SEQ IDNO:27) K₆-NH₂ 1840 [Leu10, Ala11, Ala24]hGLP- 2(1-33)-NH₂H-HGDGSFSDELATILDNLAARDFIAWLIQTKITD- (SEQ ID NO:28) NH₂ 1841 [Leu10,Ala11, Ala24]hGLP- 2(1-30)-NH₂ H-HGDGSFSDELATILDNLAARDFIAWLIQTK-NH₂ (SEQID NO:29) 1842 [Thr7, Ser8, Leu10, Lys11, Ala24]hGLP-2(1-30)-NH₂H-HGDGSFTSELKTILDNLAARDFIAWLIQTK-NH₂ (SEQ ID NO:30) 1843 [Thr7, Leu10,Ala11, Ala24] hGLP-2(1-30)-NH₂ H-HGDGSFTDELATILDNLAARDFIAWLIQTK-NH₂ (SEQID NO:31) 1844 [Gly2, Glu3, Thr5, Ser8, 11, Leu10, Ala16, 24,28]hGLP-2(1-33)- (Lys)₆-NH₂ H-HGEGTFSSELSTILDALAARDFIAWLIATKITD (SEQ IDNO:32) K₆-NH₂ 1845 [Gly2, Glu3, Thr5, Leu10, Ser11, Ala16, 24,28]hGLP-2(1-33)- (Lys)₆-NH₂ H-HGEGTFSDELSTILDALAARDFIAWLIATKITD (SEQ IDNO:33) K₆-NH₂ 1846 [Gly2, Glu3, Ser8, 11, Leu10, Ala16, 24,28]hGLP-2(1-33)-(Lys)₆- NH₂ H-HGEGSFSSELSTILDALAARDFIAWLIATKITD (SEQ IDNO:34) K₆NH₂ 1847 [Gly2, Glu3, Leu10, Ser11, Ala16, 24,28]hGLP-2(1-33)-(Lys)₆- NH₂ H-HGEGSFSDELSTILDALAARDFIAWLIATKITD (SEQ IDNO:35) K₆-NH₂ 1848 [Gly2, Glu3, Thr5, Ser8, Leu10, Ala11, 16, 24,28]hGLP-2(1-33)- (Lys)₆-NH₂ H-HGEGTFSSELATILDALAARDFIAWLIATKITD (SEQ IDNO:36) K₆-NH₂ 1849 [Gly2, Glu3, Ser8, Leu10, Ala11, 16, 24,28]hGLP-2(1-33)- (Lys)₆-NH₂ H-HGEGSFSSELATILDALAARDFIAWLIATKITD (SEQ IDNO:37) K₆-NH₂ 1850 [Gly2, Glu3, Leu10, Lys11, Ala16, 24,28]hGLP-2(1-33)- (Lys)₆-NH₂ H-HGEGSFSDELKTILDALAARDFIAWLIATKITD (SEQ IDNO:38) K₆-NH₂ 1851 [Gly2, Glu3, Thr5, Leu10, Lys11, Ala16, 24,28]hGLP-2(1-33)- (Lys)₆-NH₂ H-HGEGTFSDELKTILDALAARDFIAWLIATKITD (SEQ IDNO:39) K₆-NH₂ 1852 [Gly2, Glu3, Thr5, Ser8, Leu10, Lys11, Ala16, 24,28]hGLP-2(1-33)- NH₂ H-HGEGTFSSELKTILDALAARDFIAWLIATKITD (SEQ ID NO:40)K₆-NH₂ 1853 [Gly2, Glu3, Thr5, Ser8, 11, Leu10, Ala16, 24,28]hGLP-2(1-33)- NH₂ H-HGEGTFSSELSTILDALAARDFIAWLIATKITD- (SEQ ID NO:41)NH₂ 1854 [Gly2, Glu3, Thr5, Leu10, Ser11, Ala16, 24, 28]hGLP-2(1-33)-NH₂ H-HGEGTFSDELSTILDALAARDFIAWLIATKITD- (SEQ ID NO:42) NH₂ 1855 [Gly2,Glu3, Thr5, Ser8, Leu10, Ala11, 16, 24, 28]hGLP-2(1-33)-NH₂H-HGEGSFSSELSTILDALAARDFIAWLIATKITD- (SEQ ID NO:43) NH₂ 1856 [Gly2,Glu3, Ser8, 11, Leu10, Ala16, 24, 28]hGLP-2(1-33)-NH₂H-HGEGSFSDELSTILDALAARDFIAWLIATKITD- (SEQ ID NO:44) NH₂ 1857 [Gly2,Glu3, Leu10, Ser11, Ala16, 24, 28]hGLP-2(1-33)-NH₂H-HGEGTFSSELATILDALAARDFIAWLIATKITD- (SEQ ID NO:45) NH₂ 1858 (Gly2,Glu3, Ser8, Leu10, Ala11, 16, 24, 28]hGLP-2(1-33)-NH₂H-HGEGSFSSELATILDALAARDFIAWLIATKITD- (SEQ ID NO:46) NH₂ 1859 [Gly2,Glu3, Leu10, Lys11, Ala16, 24, 28]hGLP-2(1-33)-NH₂H-HGEGSFSDELKTILDALAARDFIAWLIATKITD- (SEQ ID NO:47) NH₂ 1860 [Gly2,Glu3, Thr5, Leu10, Lys11, Ala16, 24, 28]hGLP-2(1-33)- NH₂H-HGEGTFSDELKTILDALAARDFIAWLIATKITD- (SEQ ID NO:48) NH₂ 1861 (Gly2,Glu3, Thr5, Ser8, Leu10, Lys11, Ala16, 24, 28]hGLP-2(1-33)- NH₂HGEGTFSSELKTILDALAARDFIAWLIATKITD (SEQ ID NO:49)

Particularly preferred compounds of the present invention includecompounds 1834 (SEQ ID NO:24), 1846 (SEQ ID NO:34), 1847 (SEQ ID NO:35),1848 (SEQ ID NO:36), 1849 (SEQ ID NO:37), 1855 (SEQ ID NO:43), 1857 (SEQID NO:45), and 1858 (SEQ ID NO:46).

1834 H-HGDGSFTSELATILDNLAARDFIAWLIQTK-NH₂ (SEQ ID NO:24) 1846H-HGEGSFSSELSTILDALAARDFIAWLIATKITDK₆NH₂ (SEQ ID NO:34) 1847H-HGEGSFSDELSTILDALAARDFIAWLIATKITDK₆-NH₂ (SEQ ID NO:35) 1848H-HGEGTFSSELATILDALAARDFIAWLIATKITDK₆-NH₂ (SEQ ID NO:36) 1849H-HGEGSFSSELATILDALAARDFIAWLIATKITDK₆-NH₂ (SEQ ID NO:37) 1855H-HGEGSFSSELSTILDALAARDFIAWLIATKITD-NH₂ (SEQ ID NO:43) 1857H-HGEGTFSSELATILDALAARDFIAWLIATKITD-NH₂ (SEQ ID NO:45) 1858H-HGEGSFSSELATILDALAARDFIAWLIATKITD-NH₂ (SEQ ID NO:46)Stability Studies

The skilled person will be able to design appropriate methods (e.g.quantitative methods) for detection of degradation products of GLP-2analogues, e.g. based on those described below. Degradation may occur asoxidation, hydrolysis and deamidation, depending on the identity andposition of the amino acids in any given GLP-2 analogue, and conditionsas pH, solution and temperature. The compounds can be ranked accordingto chemical stability, when the compounds are incubated under stressedconditions (i.e. conditions likely to cause degradation) andsubsequently analysed for content of remaining intact peptide. Inaddition, the knowledge gained about major degradation products obtainedunder stressed conditions will be important for any later analyticalmethod development.

Quantitative Assays to Detect GLP Analogues

The skilled person will also be capable of designing methods (e.g.quantitative methods) for detection of GLP analogues in complexenvironments or solutions (e.g. plasma, urine, tissue homogenates, cellhomogenates, saliva or similar) to investigate the absorption,distribution, metabolism and excretion of the GLP analogues afteradministration to mammals or as part of functional studies of in vitrocell systems.

In one embodiment, a quantitative assay can be based on antibodiesraised against the GLP analogues or fragments thereof. The antibodiesobtained from the immunized animals can be used for quantitative assays.In one example a direct sandwich ELISA can be prepared using a firstantibody with affinity of one part of the molecule immobilized in amulti-well plate. The sample is then applied to the wells and the GLPanalogue is captured by the first antibody. The captured GLP analogue isthen recognized by a second antibody with affinity for another part ofthe GLP analogue. The second antibody can be labeled with an enzyme(horseradish peroxidase, alkaline phosphatase or beta-galactosidase) ora radioisotope. The amount of captured GLP analogue can then be detectedby addition of a colorimetric substrate or direct counting ofradio-emission or by scintillation. Alternatively, the amount ofcaptured GLP analogue can be detected indirectly by addition of alabeled antibody with affinity for the second antibody. Theconcentration in the sample can be estimated from the response obtainedfrom an external standard curve containing known amounts of GLPanalogue. Alternatively, the antibodies can be used to prepare a directcompetitive immuno assay, where an antibody specific for the GLPanalogue is immobilized on a multi-well plate and the sample incubatedin the wells with a predefined fixed concentration of labeled GLPanalogue. The label can be an enzyme, a fluorophore, a radioisotope orbiotin and detected using, for example, substrates (e.g. colorimetric,fluorometric or chemiluminiscent) specific for the enzymes,scintillation or avidin linked to an enzyme followed by detection asdescribed above. The amount of bound labeled GLP analogue can bedetected by an appropriate method and the concentration of GLP analoguepresent in the sample derived from the response obtained from anexternal standard curve as described above.

In another embodiment, a quantitative assay can be based on liquidchromatography tandem mass spectroscopy methodology. In such a set up,the response from a fragment specific for the GLP analogue to be studiedis monitored upon fragmentation of the parent compound induced bycollision with an inert gas (He or Ar). Prior to fragmentation thesample components can be separated by reversed phase chromatography orthe sample can be injected directly in the mass spectrometer. Ifsuitable the sample can be subjected to pretreatment (i.e., addition ofprotease inhibitors, protein precipitation, solid phase extraction,immuno-affinity extraction, etc. The concentration of GLP analoguepresent in the sample derived from the response obtained from anexternal standard curve as described above, potentially after correctionof the response using an internal standard similar to the GLP analogueto be studied.

Generation of Specific Antibodies

Specific antibodies against the GLP analogues or fragments thereof canbe induced in mammals and purified from the serum. The GLP analogues orfragments can either be used directly with an adjuvant to immunizerabbits, mice or other mammals, or the GLP analogues or fragmentsthereof can be chemically linked to a carrier molecule (i.e., keyholelimpet hemocyanin, ovalbumin, albumin etc.) and injected with anadjuvant. The injections can be repeated with 2-4 weeks intervals forextended periods to improve the affinity and selectivity of theantibodies. Polyclonal antibodies can be harvested directly from theserum. To obtain monoclonal antibodies, B cells isolated from immunizedanimals, preferably mice, should be fused with tumor cells to formantibody producing hybridomas. Screening and selection of theappropriate clones and antibodies can be performed using eitherimmobilized GLP analogues or peptides thereof followed by detection withlabeled anti-antibodies. Alternatively the screening and selection couldbe based on immobilized antibodies followed by detection with labeledGLP analogues or fragments thereof. In all cases, the label could be aradioisotope, an enzyme, a fluorophore or biotin and detected using, forexample, substrates (e.g. colorimetric, fluorometric orchemiluminiscent) specific for the enzymes, scintillation or avidinlinked to an enzyme followed by detection as described.

Synthesis of GLP-2 Analogues

It is preferred to synthesize the analogues of the invention by means ofsolid phase or liquid phase peptide synthesis. In this context,reference is given to WO 98/11125 and, amongst many others, Fields, G Bet al., 2002, “Principles and practice of solid-phase peptidesynthesis”. In: Synthetic Peptides (2nd Edition) and the Examplesherein.

Thus the GLP-2 analogues may be synthesized in a number of waysincluding for example, a method which comprises:

(a) synthesizing the peptide by means of solid phase or liquid phasepeptide synthesis and recovering the synthetic peptide thus obtained; or

(b) when the peptide is constituted by naturally occurring amino acids,expressing a nucleic acid construct that encodes the peptide in a hostcell and recovering the expression product from the host cell culture;or

(c) when the peptide is constituted by naturally occurring amino acids,effecting cell-free in vitro expression of a nucleic acid construct thatencodes the peptide and recovering the expression product; or

a combination of methods of (a), (b), and (c) to obtain fragments of thepeptide, subsequently ligating the fragments to obtain the peptide, andrecovering the peptide.

Thus, for some analogues of the invention it may be advantageous toexploit genetic engineering techniques. This may be the case when thepeptide is sufficiently large (or produced as a fusion construct) andwhen the peptide only includes naturally occurring amino acids that canbe translated from RNA in living organisms.

For the purpose of recombinant gene technology nucleic acid fragmentsencoding the peptides of the invention are important chemical products.Hence, a further aspect of the present invention provides a nucleic acidmolecule comprising a nucleic acid sequence encoding a GLP-2 analogue ofthe invention, where the peptide preferably is comprised by naturallyoccurring amino acids. The nucleic acid fragments of the invention areeither DNA or RNA fragments.

The nucleic acid fragments of the invention will normally be inserted insuitable vectors to form cloning or expression vectors carrying thenucleic acid fragments of the invention; such novel vectors are alsopart of the invention. Details concerning the construction of thesevectors of the invention will be discussed in context of transformedcells and microorganisms below. The vectors can, depending on purposeand type of application, be in the form of plasmids, phages, cosmids,mini-chromosomes, or virus, but also naked DNA which is only expressedtransiently in certain cells is an important vector. Preferred cloningand expression vectors (plasmid vectors) of the invention are capable ofautonomous replication, thereby enabling high copy-numbers for thepurposes of high-level expression or high-level replication forsubsequent cloning.

The general outline of a vector of the invention comprises the followingfeatures in the 5′→3′ direction and in operable linkage: a promoter fordriving expression of the nucleic acid fragment of the invention,optionally a nucleic acid sequence encoding a leader peptide enablingsecretion (to the extracellular phase or, where applicable, into theperiplasma) of or a leader peptide for multiple use e.g. combinedsecretion, purification tag and enzymatic trimming to correct peptide orintegration into the membrane of the polypeptide fragment, the nucleicacid fragment encoding the peptide of the invention, and optionally anucleic acid sequence encoding a terminator. When operating withexpression vectors in producer strains or cell-lines it is for thepurposes of genetic stability of the transformed cell preferred that thevector when introduced into a host cell is integrated in the host cellgenome.

The vectors of the invention are used to transform host cells to producethe modified peptide of the invention. Such transformed cells, which arealso part of the invention, can be cultured cells or cell lines used forpropagation of the nucleic acid fragments and vectors of the invention,or used for recombinant production of the peptides of the invention.

Preferred transformed cells of the invention are micro-organisms such asbacteria (such as the species Escherichia (e.g. E. coli), Bacillus (e.g.Bacillus subtilis), Salmonella, or Mycobacterium (preferablynon-pathogenic, e.g. M. bovis BCG), yeasts (such as Saccharomycescerevisiae), and protozoans. Alternatively, the transformed cells arederived from a multicellular organism, i.e. it may be fungal cell, aninsect cell, a plant cell, or a mammalian cell. Also cells derived froma human being are interesting, cf. the discussion of cell lines andvectors below. For the purposes of cloning and/or optimised expressionit is preferred that the transformed cell is capable of replicating thenucleic acid fragment of the invention. Cells expressing the nucleicfragment are preferred useful embodiments of the invention; they can beused for small-scale or large-scale preparation of the peptides of theinvention.

When producing the peptide of the invention by means of transformedcells, it is convenient, although far from essential, that theexpression product is either exported out into the culture medium orcarried on the surface of the transformed cell.

When an effective producer cell has been identified it is preferred, onthe basis thereof, to establish a stable cell line which carries thevector of the invention and which expresses the nucleic acid fragmentencoding the peptide. Preferably, this stable cell line secretes orcarries the peptide of the invention, thereby facilitating purificationthereof.

In general, plasmid vectors containing replicon and control sequences,which are derived from species compatible with the host cell, are usedin connection with the hosts. The vector ordinarily carries areplication site, as well as marking sequences, which are capable ofproviding phenotypic selection in transformed cells. For example, E.coli is typically transformed using pBR322 (but numerous other usefulplasmids exist) a plasmid derived from an E. coli species (see, e.g.,Bolivar et al., 1977). The pBR322 plasmid contains genes for ampicillinand tetracycline resistance and thus provides easy means for identifyingtransformed cells. The pBR plasmid, or other microbial plasmid or phagemust also contain, or be modified to contain promoters, which can beused by the prokaryotic microorganism for expression.

Those promoters most commonly used in prokaryotic recombinant DNAconstruction include the β-lactamase (penicillinase) and lactosepromoter systems (Chang et al., 1978; Itakura et al., 1977; Goeddel etal., 1979) and a tryptophan (trp) promoter system (Goeddel et al., 1979;EP 0 036 776 A). While these are the most commonly used, other microbialpromoters have been discovered and utilized, and details concerningtheir nucleotide sequences have been published, enabling a skilledworker to ligate them functionally with plasmid vectors (Siebwenlist etal., 1980).

In addition to prokaryotes, eukaryotic microbes, such as yeast culturesmay also be used, and also here the promoter should be capable ofdriving expression. Saccharomyces cerevisiae, or common baker's yeast isthe most commonly used among eukaryotic microorganisms, although anumber of other strains are commonly available. For expression inSaccharomyces, the plasmid YRp7, for example, is commonly used(Stinchcomb et al., 1979; Kingsman et al., 1979; Tschemper et al.,1980). This plasmid already contains the trpl gene which provides aselection marker for a mutant strain of yeast lacking the ability togrow in tryptophan for example ATCC No. 44076 or PEP4-1 (Jones, 1977).The presence of the trpl lesion as a characteristic of the yeast hostcell genome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzman et al., 1980) or other glycolyticenzymes (Hess et al., 1968; Holland et al., 1978), such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also ligated into the expression vector 3′ of the sequencedesired to be expressed to provide polyadenylation of the mRNA andtermination.

Other promoters, which have the additional advantage of transcriptioncontrolled by growth conditions are the promoter region for alcoholdehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymesassociated with nitrogen metabolism, and the aforementionedglyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible formaltose and galactose utilization. Any plasmid vector containing ayeast-compatible promoter, origin of replication and terminationsequences is suitable.

In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. However, interest has been greatest in vertebrate cells, andpropagation of vertebrate in culture (tissue culture) has become aroutine procedure in recent years (Tissue Culture, 1973). Examples ofsuch useful host cell lines are VERO and HeLa cells, Chinese hamsterovary (CHO) cell lines, and W138, BHK, COS-7 293, Spodoptera frugiperda(SF) cells (commercially available as complete expression systems fromi.a. Protein Sciences, 1000 Research Parkway, Meriden, Conn. 06450,U.S.A. and from Invitrogen), the D. melanogaster cell line S₂ availablefrom Invitrogen, PO Box 2312, 9704 CH Groningen, The Netherlands, andMDCK cell lines.

Expression vectors for such cells ordinarily include (if necessary) anorigin of replication, a promoter located in front of the gene to beexpressed, along with any necessary ribosome binding sites, RNA splicesites, polyadenylation site, and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expressionvectors are often provided by viral material. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, and most frequentlySimian Virus 40 (SV40). The early and late promoters of SV40 virus areparticularly useful because both are obtained easily from the virus as afragment, which also contains the SV40 viral origin of replication(Fiers et al., 1978). Smaller or larger SV40 fragments may also be used,provided there is included the approximately 250 bp sequence extendingfrom the HindIII site toward the BgII site located in the viral originof replication. Further, it is also possible, and often desirable, toutilize promoter or control sequences normally associated with thedesired gene sequence, provided such control sequences are compatiblewith the host cell systems.

An origin of replication may be provided either by construction of thevector to include an exogenous origin, such as may be derived from SV40or other viral (e.g. Polyoma, Adeno, VSV, BPV) or may be provided by thehost cell chromosomal replication mechanism. If the vector is integratedinto the host cell chromosome, the latter is often sufficient.

In order to obtain satisfactory yields in a recombinant productionprocess, it may be advantageous to prepare the analogues as fusionproteins, either by fusing the peptide to a fusion partner that canserve as an affinity tag (for ease of purification) and/or by havingmultiple repeats of the peptide. These methods require presence of asuitable cleavage site for a peptidase, but the skilled person will knowhow to tailor the underlying genetic constructs.

After recombinant preparation, the peptides of the invention can bepurified by methods generally known in the art, including multi-stepchromatography (e.g., ion-exchange, size-exclusion, and affinitychromatographic techniques).

Alternatively, peptides comprised of naturally occurring amino acids canbe prepared in vitro in cell free systems. This is especially expedientin cases where the peptides could be toxic for putative host cells.Thus, the present invention also contemplates use of cell-free in vitrotranslation/expression in order to prepare the peptides of theinvention. In this context, reference is made to commercially availablein vitro translation kits, materials, and technical documentation frome.g., Ambion Inc., 2130 Woodward, Austin, Tex. 78744-1832, USA.

Finally, the available methods can of course be combined so as toprepare e.g. semi-synthetic analogues. In such a set up, peptidefragments are prepared using at least 2 separate steps or methods,followed by ligation of the fragments to obtain the final peptideproduct.

Pharmaceutical Compositions and Administration

The GLP-2 analogues of the present invention, or salts or derivativesthereof, may be formulated as pharmaceutical compositions prepared forstorage or administration, and which comprise a therapeuticallyeffective amount of a GLP-2 peptide of the present invention, or a saltor derivative thereof, in a pharmaceutically acceptable carrier.

The therapeutically effective amount of a compound of the presentinvention will depend on the route of administration, the type of mammalbeing treated, and the physical characteristics of the specific mammalunder consideration. These factors and their relationship to determiningthis amount are well known to skilled practitioners in the medical arts.This amount and the method of administration can be tailored to achieveoptimal efficacy so as to deliver the peptide to the large intestine,but will depend on such factors as weight, diet, concurrent medicationand other factors, well known those skilled in the medical arts.

It is within the invention to provide a pharmaceutical composition,wherein the GLP-2 analogue, or a salt thereof is present in an amounteffective to treat or prevent stomach and bowel-related disorders.

Pharmaceutically acceptable salts of the compounds of the inventionhaving an acidic moiety can be formed using organic and inorganic bases.Suitable salts formed with bases include metal salts, such as alkalimetal or alkaline earth metal salts, for example sodium, potassium, ormagnesium salts; ammonia salts and organic amine salts, such as thoseformed with morpholine, thiomorpholine, piperidine, pyrrolidine, amono-, di- or tri-lower alkylamine (e.g., ethyl-tert-butyl-, diethyl-,diisopropyl-, triethyl-, tributyl- or dimethylpropylamine), or a mono-,di- or trihydroxy lower alkylamine (e.g., mono-, di- ortriethanolamine). Internal salts also may be formed. Similarly, when acompound of the present invention contains a basic moiety, salts can beformed using organic and inorganic acids. For example, salts can beformed from the following acids: acetic, propionic, lactic, citric,tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, phthalic,hydrochloric, hydrobromic, phosphoric, nitric, sulfuric,methanesulfonic, napthalenesulfonic, benzenesulfonic, toluenesulfonic,and camphorsulfonic as well as other known pharmaceutically acceptableacids. Amino acid addition salts can also be formed with amino acidssuch as lysine, glycine, or phenylalanine.

As is apparent to one skilled in the medical art, a “therapeuticallyeffective amount” of the peptides or pharmaceutical compositions of thepresent invention will vary depending upon the age, weight and mammalianspecies treated, the particular compounds employed, the particular modeof administration and the desired effects and the therapeuticindication. Because these factors and their relationship to determiningthis amount are well known in the medical arts, the determination oftherapeutically effective dosage levels, the amount necessary to achievethe desired result of preventing and/or treating the intestine andstomach related diseases described herein, as well as other medicalindications disclosed herein, will be within the ambit of the skilledperson.

As used herein, “a therapeutically effective amount” is one whichreduces symptoms of a given condition or pathology, and preferably whichnormalizes physiological responses in an individual with the conditionor pathology. Reduction of symptoms or normalization of physiologicalresponses can be determined using methods routine in the art and mayvary with a given condition or pathology. In one aspect, atherapeutically effective amount of one or more GLP-2 analogues orpharmaceutical composition comprising the one or more GLP-2 analogues isan amount which restores a measurable physiological parameter tosubstantially the same value (preferably to within +30%, more preferablyto within +20%, and still more preferably, to within 10% of the value)of the parameter in an individual without the condition or pathology.

In one embodiment of the invention administration of the compounds orpharmaceutical composition of the present invention is commenced atlower dosage levels, with dosage levels being increased until thedesired effect of preventing/treating the relevant medical indication,such as intestine and stomach related diseases is achieved. This woulddefine a therapeutically effective amount. For the peptides of thepresent invention, alone or as part of a pharmaceutical composition,such doses may be between about 0.01 mg/kg and 100 mg/kg body weight,such as between about 0.01 mg/kg and 10 mg/kg body weight, for examplebetween 10-100 μg/kg body weight.

For therapeutic use, the chosen GLP-2 analogue is formulated with acarrier that is pharmaceutically acceptable and is appropriate fordelivering the peptide by the chosen route of administration. For thepurpose of the present invention, peripheral parenteral routes includeintravenous, intramuscular, subcutaneous, and intra peritoneal routes ofadministration. Certain compounds used in the present invention may alsobe amenable to administration by the oral, rectal, nasal, or lowerrespiratory routes. These are so-called non-parenteral routes. Thepresent pharmaceutical composition comprises a GLP-2 analogue of theinvention, or a salt or derivative thereof and a pharmaceuticallyacceptable carrier. Suitable pharmaceutically acceptable carriers arethose used conventionally with peptide-based drugs, such as diluents,excipients and the like. Pharmaceutically acceptable carriers fortherapeutic use are well known in the pharmaceutical art, and aredescribed, for example, in Remington's Pharmaceutical Sciences, MackPublishing Co. (A. R. Gennaro edit. 1985). For example, sterile salineand phosphate-buffered saline at slightly acidic or physiological pH maybe used. pH buffering agents may be phosphate, citrate, acetate,tris/hydroxymethyl)aminomethane (TRIS),N-Tris(hydroxymethyl)methyl-3-aminopropanesulphonic acid (TAPS),ammonium bicarbonate, diethanolamine, histidine, which is a preferredbuffer, arginine, lysine, or acetate or mixtures thereof. Preferredbuffer ranges are pH 4-8, pH 6.5-8, more preferably pH 7-7.5.Preservatives, such as para, meta, and ortho-cresol, methyl- andpropylparaben, phenol, benzyl alcohol, sodium benzoate, benzoic acid,benzyl-benzoate, sorbic acid, propanoic acid, esters of p-hydroxybenzoicacid may be provided in the pharmaceutical composition. Stabilizers,preventing oxidation, deamidation, isomerisation, racemisation,cyclisation, peptide hydrolysis, such as e.g. ascorbic acid, methionine,tryptophane, EDTA, asparagine, lysine, arginine, glutamine and glycinemay be provided in the pharmaceutical composition. Stabilizers,preventing aggregation, fibrillation and precipitation, such as Sodiumdodecyl sulphate, polyethylene glycol, carboxymethyl cellulose,cyclodextrine may be provided in the pharmaceutical composition. Organicmodifiers for solubilization or preventing aggregation, such as ethanol,acetic acid or acetate and salts thereof may be provided in thepharmaceutical composition. Isotonicity makers such as salts e.g. sodiumchloride or most preferred carbohydrates e.g. dextrose, mannitol,lactose, trehalose, sucrose or mixtures thereof may be provided in thepharmaceutical composition.

Detergents, such as Tween 20, Tween 80, SDS, Poloxamers e.g. PluronicF-68, Pluronic F-127, may be provided in the pharmaceutical composition.Dyes and even flavoring agents may be provided in the pharmaceuticalcomposition. In another embodiment, a pharmaceutically acceptable acidaddition salt of the GLP-2 peptide analogue is provided for. Suspendingagents may be used.

Organic modifiers, such as ethanol, tertiary-buthanol, 2-propanol,ethanol, glycerol, Polyethylene glycol may be provided in thepharmaceutical formulation for lyophilization of a lyophilized product.Bulking agents and isotonicity makers such as salt e.g. sodium chloride,carbohydrates e.g. dextrose, mannitol, lactose, trehalose, sucrose ormixtures thereof, aminoacids e.g. glycine, glutamate, or excipients suchas cystein, lecithin or human serum albumin, or mixtures thereof may beprovided in the pharmaceutical composition for lyophilization.

The pharmaceutical compositions of the present invention may beformulated and used as tablets, capsules or elixirs for oraladministration; suppositories for rectal administration; preferablysterile solutions or sterile powder or suspensions for injectableadministration; and the like. The dose and method of administration canbe tailored to achieve optimal efficacy but will depend on such factorsas weight, diet, concurrent medication and other factors, which thoseskilled in the medical arts will recognize.

When administration is to be parenteral, such as intravenous andsubcutaneous, e.g., on a daily basis, injectable pharmaceuticalcompositions can be prepared in conventional forms, either as aqueoussolutions or suspensions; lyophilized, solid forms suitable forreconstitution immediately before use or suspension in liquid prior toinjection, or as emulsions.

Diluents for reconstitution of the lyophilized product may be a suitablebuffer from the list above, water, saline, dextrose, mannitol, lactose,trehalose, sucrose, lecithin, albumin, sodium glutamate, cysteinehydrochloride; or water for injection with addition of detergents, suchas Tween 20, Tween 80, poloxamers e.g. pluronic F-68 or pluronic F-127,polyethylene glycol, and or with addition of preservatives such aspara-, meta-, and ortho-cresol, methyl- and propylparaben, phenol,benzyl alcohol, sodium benzoate, benzoic acid, benzyl-benzoate, sorbicacid, propanoic acid, esters of p-hydroxybenzoic acid, and or withaddition of an organic modifier such as ethanol, acitic acid, citricacid, lactic acid or salts thereof.

In addition, if desired, the injectable pharmaceutical compositions maycontain minor amounts of non-toxic auxiliary substances, such as wettingagents, or pH buffering agents. Absorption enhancing preparations (e.g.,liposomes, detergents and organic acids) may be utilized.

In one embodiment of the invention, the compounds are formulated foradministration by infusion, e.g., when used as liquid nutritionalsupplements for patients on total parenteral nutrition therapy (forexample neonatals, or patients suffering from cachexia or anorexia), orby injection, for example subcutaneously, intraperitoneal orintravenously, and are accordingly utilized as aqueous solutions insterile and pyrogen-free form and optionally buffered to physiologicallytolerable pH, e.g., a slightly acidic or physiological pH. Formulationfor intramuscular administration may be based on solutions orsuspensions in plant oil, e.g. canola oil, corn oil or soy bean oil.These oil based formulations may be stabilized by antioxidants e.g. BHA(butylated hydroxianisole) and BHT (butylated hydroxytoluene).

Thus, the present peptide compounds may be administered in a vehicle,such as distilled water or in saline, phosphate buffered saline, 5%dextrose solutions or oils. The solubility of the GLP-2 analogue may beenhanced, if desired, by incorporating a solubility enhancer, such asdetergents and emulsifiers.

The aqueous carrier or vehicle can be supplemented for use asinjectables with an amount of gelatin that serves to depot the GLP-2analogue at or near the site of injection, for its slow release to thedesired site of action. Alternative gelling agents, such as hyaluronicacid, may also be useful as depot agents.

In one embodiment of the present invention the formulation comprises

a. L-histidine dissolved in water to obtain final concentrations of from0.5 mM to 300 mM, preferably from 3 to 200 mM, most preferably from 20to 100 mM;

b. mannitol to obtain up to 350 mM, preferably from 30 to 300 mM, mostpreferably from 100 mM to 230 mM; and

c. acetic acid to obtain up to 200 mM, preferably from 0.05 to 100 mM,most preferably from 0.5 to 50 mM into solution.

Appropriate amount of therapeutic compound is added to obtainconcentrations of from 1 to 100 mg/mL, preferably from 5 to 50 mg/mL,most preferably from 10 to 30 mg/mL.

pH are adjusted to final pH at from 4 to 8, preferably from 6.5 to 7.5,most preferably from 6.7 to 7.3. The resulting solution is adjusted totarget weight, sterile filtered and dispensed into appropriate aliquotsin vials for pharmaceutical use. The formulation is further processedaccording to a liquid product or to a lyophilised product.

In another embodiment of the present invention the formulation comprises

-   a. L-histidine dissolved in water to obtain final concentrations of    from 0.5 mM to 300 mM, preferably from 3 to 200 mM, most preferably    from 20 to 100 mM L-histidine;-   b. L-Arginine to obtain up to 200 mM, preferably from 0.5 to 100 mM,    most preferably from 5 to 50 mM;-   c. mannitol to obtain up to 350 mM, preferably from 30 to 300 mM,    most preferably from 100 mM to 230 mM; and-   d. acetic acid to obtain up to 200 mM, preferably from 0.05 to 100    mM, most preferably from 0.5 to 50 mM into solution.

Appropriate amount of therapeutic compound is added to obtainconcentrations of from 1 to 100 mg/mL, preferably from 5 to 50 mg/mL,most preferably from 10 to 30 mg/mL.

pH are adjusted to final pH at from 4 to 8, preferably from 6.5 to 7.5,most preferably from 6.7 to 7.3. The resulting solution is adjusted totarget weight, sterile filtered and dispensed into appropriate aliquotsin vials for pharmaceutical use. The formulation is further processedaccording to a liquid product or to a lyophilised product.

In still another embodiment of the present invention the formulationcomprises

-   a. L-histidine dissolved in water to obtain final concentrations of    up to 200 mM, preferably from 3 to 100 mM, most preferably from 5 to    50 mM L-histidine;-   b. L-Arginine to obtain up to 200 mM, preferably from 0.5 to 100 mM,    most preferably from 5 to 50 mM;-   c. mannitol to obtain up to 350 mM, preferably from 30 to 300 mM,    most preferably from 100 mM to 230 mM; and-   d. acetic acid to obtain up to 200 mM, preferably from 0.05 to 100    mM, most preferably from 0.5 to 50 mM into solution.

Appropriate amount of therapeutic compound is added to obtainconcentrations of from 1 to 100 mg/mL, preferably from 5 to 50 mg/mL,most preferably from 10 to 30 mg/mL.

pH are adjusted to final pH at from 4 to 8, preferably from 6.5 to 7.5,most preferably from 6.7 to 7.3. The resulting solution is adjusted totarget weight, sterile filtered and dispensed into appropriate aliquotsin vials for pharmaceutical use. The formulation is further processedaccording to a liquid product or to a lyophilised product.

In yet another embodiment of the present invention the formulationcomprises

-   a. L-histidine dissolved in water to obtain final concentrations of    from 0.5 to 300 mM, preferably from 3 to 200 mM, most preferably    from 20 to 100 mM L-histidine;-   b. L-Arginine to obtain up to 200 mM, preferably from 0.5 to 100 mM,    most preferably from 5 to 50 mM;-   c. mannitol to obtain up to 350 mM, preferably from 30 to 300 mM,    most preferably from 100 mM to 230 mM; and-   d. acetic acid to obtain up to 200 mM, preferably from 0.05 to 100    mM, most preferably from 0.5 to 50 mM into solution.

Appropriate amount of therapeutic compound is added to obtainconcentrations of from 1 to 100 mg/mL, preferably from 5 to 50 mg/mL,most preferably from 10 to 30 mg/mL.

pH are adjusted to final pH at from 4 to 8, preferably from 6.5 to 7.5,most preferably from 6.7 to 7.3. The resulting solution is adjusted totarget weight, sterile filtered and dispensed into appropriate aliquotsin vials for pharmaceutical use. The formulation is further processedaccording to a liquid product or to a lyophilised product.

In yet another embodiment of the present invention the formulationcomprises

-   a. L-histidine dissolved in water to obtain final concentrations of    from up to 200 mM, preferably from 3 to 100 mM, most preferably from    5 to 50 mM L-histidine;-   b. L-Arginine to obtain up to 200 mM, preferably from 0.5 to 100 mM,    most preferably from 5 to 50 mM;-   c. mannitol to obtain up to 350 mM, preferably from 30 to 300 mM,    most preferably from 100 mM to 230 mM; and-   d. acetic acid to obtain up to 200 mM, preferably from 0.05 to 100    mM, most preferably from 0.5 to 50 mM into solution.

Appropriate amount of therapeutic compound is added to obtainconcentrations of from 1 to 100 mg/mL, preferably from 5 to 50 mg/mL,most preferably from 10 to 30 mg/mL.

pH are adjusted to final pH at from 4 to 8, preferably from 6.5 to 7.5,most preferably from 6.7 to 7.3. The resulting solution is adjusted totarget weight, sterile filtered and dispensed into appropriate aliquotsin vials for pharmaceutical use. The formulation is further processedaccording to a liquid product or to a lyophilised product.

In yet another embodiment of the present invention the formulationcomprises

-   a. N-acetate dissolved in water to obtain final concentrations of    from up to 200 mM, preferably from 0.5 to 100 mM, most preferably    from 5 to 50 mM L-histidine;-   b. mannitol to obtain up to 350 mM, preferably from 30 to 300 mM,    most preferably from 100 mM to 230 mM.

Appropriate amount of therapeutic compound is added to obtainconcentrations of from 1 to 100 mg/mL, preferably from 5 to 50 mg/mL,most preferably from 10 to 30 mg/mL.

pH are adjusted to final pH at from 4 to 8, preferably from 6.5 to 7.5,most preferably from 6.7 to 7.3. The resulting solution is adjusted totarget weight, sterile filtered and dispensed into appropriate aliquotsin vials for pharmaceutical use. The formulation is further processedaccording to a liquid product or to a lyophilised product

The GLP-2 analogues of the invention may also be formulated as a slowrelease implantation device for extended and sustained administration ofthe GLP-2 peptide analogue. Such sustained release formulations may bein the form of a patch positioned externally on the body. Examples ofsustained release formulations include composites of biocompatiblepolymers, such as poly(lactic acid), poly(lactic-co-glycolic acid),methylcellulose, hyaluronic acid, sialic acid, silicate, collagen,liposomes and the like. Sustained release formulations may be ofparticular interest when it is desirable to provide a high localconcentration of a GLP-2 analogue of the invention.

The GLP-2 analogue may be utilized in the form of a sterile-filled vialor ampoule containing an intestinotrophic amount of the peptide, ineither unit dose or multi-dose amounts. The vial or ampoule may containthe GLP-2 analogue and the desired carrier, as an administration readyformulation. Alternatively, the vial or ampoule may contain the GLP-2peptide in a form, such as a lyophilized form, suitable forreconstitution in a suitable carrier, such as sterile water orphosphate-buffered saline.

As an alternative to injectable formulations, the GLP-2 analogue may beformulated for administration by other routes. Oral dosage forms, suchas tablets, capsules and the like, can be formulated in accordance withstandard pharmaceutical practice. According to the present invention,the GLP-2 analogue is administered to treat individuals that wouldbenefit from growth of small bowel tissue.

Nasal dosage forms can be formulated with addition of enhancers, such asChitosan or detergents such as Tween 20, Tween 80, Poloxamers, e.g.,Pluronic F-68, Pluronic F-127; Brij 35, Brij 72, cremophor EL.

The peptide compounds of the present invention may be used alone, or incombination with compounds having an anti-inflammatory effect. Withoutbeing bound by theory, it is envisioned that such combination treatmentmay enforce the beneficial treatment effects of the present peptideanalogues.

The therapeutic dosing and regimen most appropriate for patienttreatment will of course vary with the disease or condition to betreated, and according to the patient's weight and other parameters.Without wishing to be bound by any particular theory, it is expectedthat doses, in the μg/kg range, and shorter or longer duration orfrequency of treatment may produce therapeutically useful results, suchas a statistically significant increase particularly in small bowelmass. In some instances, the therapeutic regimen may include theadministration of maintenance doses appropriate for preventing tissueregression that occurs following cessation of initial treatment. Thedosage sizes and dosing regimen most appropriate for human use may beguided by the results obtained by the present invention, and may beconfirmed in properly designed clinical trials.

An effective dosage and treatment protocol may be determined byconventional means, starting with a low dose in laboratory animals andthen increasing the dosage while monitoring the effects, andsystematically varying the dosage regimen as well. Numerous factors maybe taken into consideration by a clinician when determining an optimaldosage for a given subject. Such considerations are known to the skilledperson.

A human dose of a GLP-2 peptide according to the invention may in oneembodiment be from about 10 μg/kg body weight/day to about 10 mg/kg/day,preferably from about 50 μg/kg/day to about 5 mg/kg/day, and mostpreferably about 100 μg/kg/day to 1 mg/kg/day.

Medical Conditions

The peptides of the present invention are useful as a pharmaceuticalagent for preventing or treating an individual suffering fromgastro-intestinal disorders, including the upper gastrointestinal tractof the oesophagus by administering an effective amount of a GLP-2analogue, or a salt thereof as described herein. The stomach andintestinal-related disorders include ulcers of any aetiology (e.g.,peptid ulcers, drug-induced ulcers, ulcers related to infections orother pathogens), digestion disorders, malabsorption syndromes,short-bowel syndrome, cul-de-sac syndrome, inflammatory bowel disease,celiac sprue (for example arising from gluten induced enteropathy orceliac disease), tropical sprue, hypogammaglobulinemic sprue, enteritis,ulcerative colitis, small intestine damage and Chemotherapy Induceddiarrhea/mucositis (CID).

As mentioned above in general, individuals who would benefit fromincreased small intestinal mass and consequent and/or maintainance ofnormal small intestine mucosal structure and function are candidates fortreatment with the present GLP-2 analogues. Particular conditions thatmay be treated with GLP-2 analogue include the various forms of sprueincluding celiac sprue which results from a toxic reaction toalpha-gliadin from heat and may be a result of gluten-inducedenteropathy or celiac disease, and is marked by a significant loss ofvillae of the small bowel; tropical sprue which results from infectionand is marked by partial flattening of the villae; hypogammaglobulinemicsprue which is observed commonly in patients with common variableimmunodeficiency or hypogammaglobulinemia and is marked by significantdecrease in villus height. The therapeutic efficacy of the GLP-2analogue treatment may be monitored by enteric biopsy to examine thevillus morphology, by biochemical assessment of nutrient absorption, bypatient weight gain, or by amelioration of the symptoms associated withthese conditions.

Other conditions that may be treated with the GLP-2 analogues of theinvention, or for which the GLP-2 analogues may be usefulprophylactically, include in addition to the above mentioned radiationenteritis, infectious or post-infectious enteritis, and small intestinaldamage due to cancer-chemotherapeutic or toxic agents.

The GLP-2 analogues may also be used for the treatment of malnutrition,for example cachexia and anorexia.

A particular embodiment the invention is concerned with using thepresent peptides for the prevention and/or treatment of intestinaldamage and dysfunction. Such damage and dysfunction is a well-known sideeffect of cancer-chemotherapy treatment. Chemotherapy administration isfrequently associated with unwanted side effects related to thegastronintestinal system such as mucositis, diarrhoea, bacterialtranslocation, malabsorption, abdominal cramping, gastrointestinalbleeding and vomiting. These side effects are clinical consequences ofthe structural and functional damage of the intestinal epithelium andfrequently make it necessary to decrease the dose and frequency ofchemotherapy. Administration of the present GLP-2 peptide agonists mayenhance trophic effect in the intestinal crypts and rapidly provide newcells to replace the damaged intestinal epithelium followingchemotherapy. The ultimate goal achieved by administering the presentpeptides is to reduce the morbidity related to gastrointestinal damageof patients undergoing chemotherapy treatment while creating the mostoptimal chemotherapy regime for the treatment of cancer. Concomitantprophylactic or therapeutic treatment may be provided in accordance withthe present invention to patients undergoing or about to undergoradiation therapy.

The stem cells of the small intestinal mucosa are particularlysusceptible to the cytotoxic effects of chemotherapy due to their rapidrate of proliferation (Keefe et al., Gut 2000; 47: 632-7).Chemotherapy-induced damage to the small intestinal mucosa is clinicallyoften referred to as gastrointestinal mucositis and is characterized byabsorptive and barrier impairments of the small intestine. For example,it has been shown that, the broadly used chemotherapeutic agents, 5-FU,irinotecan and methothrexate increase apoptosis leading to villusatrophy and crypt hypoplasia in the small intestine of rodents (Keefe etal., Gut 47: 632-7, 2000; Gibson et al., J Gastroenterol Hepatol. Sep;18(9):1095-1100, 2003; Tamaki et al., J Int Med Res. 31(1):6-16, 2003).Chemotherapeutic agents have been shown to increase apoptosis inintestinal crypts at 24 hours after administration and subsequently todecrease villus area, crypt length, mitotic count per crypt, andenterocyte height three days after chemotherapy in humans (Keefe et al.,Gut 2000; 47: 632-7). Thus, structural changes within the smallintestine directly lead to intestinal dysfunction and in some casesdiarrhea.

Gastrointestinal mucositis after cancer chemotherapy is an increasingproblem that is essentially untreatable once established, although itgradually remits. Studies conducted with the commonly used cytostaticcancer drugs 5-FU and irinotecan have demonstrated that effectivechemotherapy with these drugs predominantly affects structural integrityand function of the small intestine while the colon is less sensitiveand mainly responds with increased mucus formation (Gibson et al., JGastroenterol Hepatol. Sep; 18(9):1095-1100, 2003; Tamaki et al., J IntMed Res. 31(1):6-16, 2003).

The novel GLP-2 analogues of the present invention may be useful in theprevention and/or treatment of gastrointestinal injury and side effectsof chemotherapeutic agents. This potentially important therapeuticapplication may apply to currently used chemotherapeutic agents such asbut not limited to: 5-FU, Altretamine, Bleomycin, Busulfan,Capecitabine, Carboplatin, Carmustine, Chlorambucil, Cisplatin,Cladribine, Crisantaspase, Cyclophosphamide, Cytarabine, Dacarbazine,Dactinomycin, Daunorubicin, Docetaxel, Doxorubicin, Epirubicin,Etoposide, Fludarabine, Fluorouracil, Gemcitabine, Hydroxycarbamide,Idarubicin, Ifosfamide, Irinotecan, Liposomal doxorubicin, Leucovorin,Lomustine, Melphalan, Mercaptopurine, Mesna, Methotrexate, Mitomycin,Mitoxantrone, Oxaliplatin, Paclitaxel, Pemetrexed, Pentostatin,Procarbazine, Raltitrexed, Streptozocin, Tegafur-uracil, Temozolomide,Thiotepa, Tioguanine/Thioguanine, Topotecan, Treosulfan, Vinblastine,Vincristine, Vindesine, Vinorelbine, Bleomycin, Busulfan, Capecitabine,Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cladribine,Crisantaspase, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin,Daunorubicin, Docetaxel, Doxorubicin, Epirubicin, Etoposide,Fludarabine, Fluorouracil, Gemcitabine, Hydroxycarbamide, Idarubicin,Ifosfamide, Irinotecan, Liposomal doxorubicin, Leucovorin, Lomustine,Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitoxantrone,Oxaliplatin, Paclitaxel, Pemetrexed, Pentostatin, Procarbazine,Raltitrexed, Streptozocin, Tegafur-uracil, Temozolomide, Thiotepa,Tioguanine/Thioguanine, Topotecan, Treosulfan, Vinblastine, Vincristine,Vindesine, and Vinorelbine.

It is envisioned that the present peptides may be employed in a methodof treating neo-natals by administering an effective amount of a GLP-2analogue, or a salt thereof. Complications with feeding neonatals due tothe lack of development of the intestine may be overcome by using thepresent peptide agonists.

In another embodiment the invention describes a method of treatingDPP-IV (dipeptidylpeptidase-IV) mediated conditions by administering toa patient in need thereof an effective amount of a GLP-2 analogue, or asalt thereof. Such diseases include conditions in which the DPP-IVenzyme is over expressed.

The pharmaceutical composition may in one embodiment be formulated tocause slow release of said GLP-2 analogue, or a salt or derivativethereof as described above.

It is envisaged that the present peptides may be employed in a method oftreating neo-natals by administering an effective amount of a GLP-2analogue, or a salt thereof. Complications with feeding neonatals due tothe lack of development of the intestine may be overcome by using thepresent peptide agonists.

In another embodiment the invention describes a method of treatingDPP-IV (dipeptidylpeptidase-IV) mediated conditions by administering toa patient in need thereof an effective amount of a GLP-2 analogue, or asalt thereof. Such diseases include conditions in which the DPP-IVenzyme is over expressed.

EXAMPLES

The following examples are provided to illustrate preferred aspects ofthe invention and are not intended to limit the scope of the invention.

General Peptide Synthesis

Apparatus and Synthetic Strategy

Peptides were synthesized batchwise in a polyethylene vessel equippedwith a polypropylene filter for filtration using9-fluorenylmethyloxycarbonyl (Fmoc) as N-α-amino protecting group andsuitable common protection groups for side-chain functionalities.

Solvents

Solvent DMF (N,N-dimethylformamide, Riedel de-Häen, Germany) waspurified by passing through a column packed with a strong cationexchange resin (Lewatit S 100 MB/H strong acid, Bayer AG Leverkusen,Germany) and analyzed for free amines prior to use by addition of3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (Dhbt-OH) giving rise toa yellow color (Dhbt-O-anion) if free amines are present. Solvent DCM(dichloromethane, analytical grade, Riedel de-Häen, Germany) was useddirectly without purification. Acetonitril (HPLC-grade, Lab-Scan, DublinIreland) was used directly without purification.

Amino Acids

Fmoc-protected amino acids were purchased from Advanced ChemTech (ACT)in suitable side-chain protected forms.

Coupling Reagents

Coupling reagent diisopropylcarbodiimide (DIC) was purchased from Riedelde-Häen, Germany.

Solid Supports

Peptides were synthesized on TentaGel S resins 0.22-0.31 mmol/g.TentaGel S-Ram, TentaGel S RAM-Lys(Boc)Fmoc (Rapp polymere, Germany)were used in cases where a C-terminal amidated peptide was preferred,while TentaGel S PHB, TentaGel S PHB Lys(Boc)Fmoc were used when aC-terminal free carboxylic acid was preferred.

Catalysts and Other Reagents

Diisopropylethylamine (DIEA) was purchased from Aldrich, Germany,piperidine and pyridine from Riedel-de Häen, Frankfurt, Germany.Ethandithiol was purchased from Riedel-de Häen, Frankfurt, Germany.3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (Dhbt-OH),1-hydroxybenzotriazole (HOBt) (HOAt) were obtained from Fluka,Switzerland. Acetic anhydride was obtained from Fluka.

Coupling Procedures

The amino acids were coupled as in situ generated HObt or HOAt estersmade from appropriate N-a-protected amino acids and HObt or HOAt bymeans of DIC in DMF. Acylations were checked by the ninhydrin testperformed at 80° C. in order to prevent Fmoc deprotection during thetest (Larsen, B. D. and Holm, A., Int. J. Peptide Protein Res. 43, 1994,1-9).

Deprotection of the N-α-Amino Protecting Group (Fmoc).

Deprotection of the Fmoc group was performed by treatment with 20%piperidine in DMF (1×5 and 1×10 min.), followed by wash with DMF (5×15ml, 5 min. each) until no yellow color could be detected after additionof Dhbt-OH to the drained DMF.

Coupling of HOBt-Esters

3 eq. N-α-amino protected amino acid was dissolved in DMF together with3 eq. HObt and 3 eq DIC and then added to the resin.

Cleavage of Peptide from Resin with Acid

Peptides were cleaved from the resins by treatment with 95%trifluoroacetic acid (TFA, Riedel-de Häen, Frankfurt, Germany)-water v/vor with 95% TFA and 5% ethandithiol v/v at r.t. for 2 h. The filteredresins were washed with 95% TFA-water and filtrates and washingsevaporated under reduced pressure. The residue washed with ether andfreeze dried from acetic acid-water. The crude freeze dried product wasanalyzed by high-performance liquid chromatography (HPLC) and identifiedby mass spectrometry (MS).

Batchwise Peptide Synthesis on TentaGel Resin (PEG-PS)

TentaGel resin (1 g, 0.23-0.24 mmol/g) was placed in a polyethylenevessel equipped with a polypropylene filter for filtration. The resinwas swelled in DMF (15 ml), and treated with 20% piperidine in DMF inorder to remove the initial Fmoc group either on the linker TentaGel SRAM or on the first amino acid on the resin TentaGel S RAM-Lys(Boc)Fmoc.The resin was drained and washed with DMF until no yellow color could bedetected after addition of Dhbt-OH to the drained DMF. The amino acidsaccording to the sequence were coupled as preformed Fmoc-protected HObtesters (3 eq.) as described above. The couplings were continued for 2 h,unless otherwise specified.

The resin was drained and washed with DMF (5×15 ml, 5 min each) in orderto remove excess reagent. All acylations were checked by the ninhydrintest as described above. After completed synthesis the peptide-resinwashed with DMF (3×15 ml, 5 min each), DCM (3×15 ml, 1 min each) andfinally diethyl ether (3×15 ml, 1 min each) and dried in vacuo. Thepeptide was cleaved from the resin as described earlier and the crudepeptide product was analysed and purified as described below

HPLC Conditions

Gradient HPLC analysis was done using a Hewlett Packard HP 1100 HPLCsystem consisting of a HP 1100 Quaternary Pump, a HP 1100 Autosampler aHP 1100 Column Thermostat and HP 1100 Multiple Wavelength Detector.Hewlett Packard Chemstation for LC software (rev. A.06.01) was used forinstrument control and data acquisition. The following columns and HPLCbuffer system was used:

Column: VYDAC 238TP5415, C-18, 5 mm, 300 Å 150×4.6 mm.

Buffers: A: 0.1% TFA in MQV; B: 0.085% TFA, 10% MQV, 90% MeCN.

Gradient: 0-1.5 min. 0% B

-   -   1.5-25 min 50% B    -   25-30 min 100% B    -   30-35 min 100% B    -   35-40 min 0% B        Flow 1, ml/min, oven temperature 40° C., UV detection: I=215 nm.        HPLC Purification of the Crude Peptide

The crude peptide products were purified PerSeptive Biosystems VISIONWorkstation. VISION 3.0 software was used for instrument control anddata acquisition. The following column and HPLC buffer system was used:

Column: Kromasil KR 100 Å, 10 mm C-8, 250×50.8 mm.

Buffer system: Buffers: A: 0.1% TFA in MQV; B: 0.085% TFA, 10% MQV, 90%MeCN.

Gradient: 0-37 min. 0-40% B

Flow 35 ml/min, UV detection: I=215 nm and 280 nm.

Mass Spectroscopy

The peptides were dissolved in super gradient methanol (Labscan, Dublin,Ireland), milli-Q water (Millipore, Bedford, Mass.) and formic acid(Merck, Damstadt, Germany) (50:50:0.1 v/v/v) to give concentrationsbetween 1 and 10 mg/ml. The peptide solutions (20 ml) were analysed inpositive polarity mode by ESI-TOF-MS using a LCT mass spectrometer(Micromass, Manchester, UK) accuracy of +/−0.1 m/z.

General Synthetic Procedure

In all syntheses dry TentaGel-S-Ram resin (1 g, 0.22-0.31 mmol/g) wasplaced in a polyethylene vessel equipped with a polypropylene filter forfiltration. The resin was swelled in DMF (15 ml), and treated with 20%piperidine in DMF to secure the presence of non-protonated amino groupson the resin. The resin was drained and washed with DMF until no yellowcolour could be detected after addition of Dhbt-OH to the drained DMF.The amino acids according to the sequence were coupled as preformedFmoc-protected HOBt esters (3 eq.) as described above. The couplingswere continued for 2 h, unless otherwise specified. The resin wasdrained and washed with DMF (5×15 ml, 5 min each) in order to removeexcess reagent. All acylations were checked by the ninhydrin testperformed at 80° C. After completed synthesis the peptide-resin washedwith DMF (3×15 ml, 5 min each), DCM (3×15 ml, 1 min each) and finallydiethyl ether (3×15 ml, 1 min each) and dried in vacuo. The peptide wasthen cleaved from the resin as described above and freeze dried. Afterpurification using preparative HPLC as described above, the peptideproduct was collected and the identity of the peptide was confirmed byES-MS. This procedure was used for the synthesis of all peptidesexemplified further below.

Compounds Synthesised

Using the above techniques compounds 1809 to 1861 (SEQ ID NOS:1-49) andreference compound 1559 (H-[Gly2]hGLP-2-OH) (SEQ ID NO:54) weresynthesised using the methods described above (Table 1).

TABLE 1 Compounds synthesized Mw Mw purity Compound # Sequence calcfound % Yield* 1559 (SEQ ID NO:54)H-HGDGSFSDEMNTILDNLAARDFINWLIQTKITD-NH2 3749.80 3749.16 95 5.4 1809 (SEQID NO:1) H-HGEGTFSDELATILDALAARDFIAWLIATKITDKKKKKK-NH2 4341.42 4341.6296 46.5 1810 (SEQ ID NO:2) H-HGEGTFSDELATILDALAARIFIAWLIATKKKKKKK-NH24010.32 4010.63 91 20 1811 (SEQ ID NO:3)H-HGDGSPSDELATILDALAARDFIAWLIATKITD-NH2 3494.8 3494.13 94 44.7 1812 (SEQID NO:4) H-HGEGSFSDELATILDNLAARDFIAWLIQTKITD-NH2 3658.86 3658.3 91 81813 (SEQ ID NO:5) H-HGDGSFSDELATILDALAARDFIAWLIATKITD-NH2 3544.82 354595 15.9 1814 (SEQ ID NO:6) H-HGDGSFSDELATILDNLAARDFIAWLIATKITD-NH23587.83 3588 92 31 1815 (SEQ ID NO:7)H-HGEGSFSDELATILDALAARDFIAWLIATKITD-NH2 3558.84 3559 95 33 1818 (SEQ IDNO:8) H-HGDGSFSSELATILDNLAARDFIAWLIQTKKKKKKK-NH2 4056.26 4056.25 92 68.31819 (SEQ ID NO:9) H-HGDGSFSDELSTILDNLAARDFIAWLIQTKKKKKKK-NH2 4100.254100.13 92 38.7 1820 (SEQ ID NO:10)H-HGDGSFTSELATILDNLAARDFIAWLIQTKKKKKKK-NH2 4070.28 4070.38 94 63.3 1821(SEQ ID NO:11) H-HGDGSFSDELKTILDNLAARDFIAWLIQTKKKKKKK-NH2 4141.32 4141.593 57.3 1822 (SEQ ID NO:12) H-HGDGSFTDELKTILDNLAARDFIAWLIQTKKKKKKK-NH24155.33 4155.13 94 72 1823 (SEQ ID NO:13)H-HGDGSFTSELKTILDNLAARDFIAWLIQTKKKKKKK-NH2 4127.34 4127.9 95 100.3 1824(SEQ ID NO:14) H-HGDGSFTDELATILDNLAARDFIAWLIQTKKKKKKK-NH2 4098.274098.25 95 33.9 1825 (SEQ ID NO:15) H-HGDGSFSSELATILDNLAARDFIAWLIQTK-NH23287.69 3287.75 92 82.0 1826 (SEQ ID NO:16)H-HGDGSFSDELNTILDNLAARDFIAWLIQTKITDKKKKKK-NH2 4456.42 4456.38 93 68.91827 (SEQ ID NO:17) H-HGDGSFTDELSTILDNLAARDFIAWLIQTKKKKKKK-NH2 4114.274114.63 93 20.2 1828 (SEQ ID NO:18)H-HGDGSFTSELSTILDNLAARDFIAWLIQTKKKKKKK-NH2 4086.27 4086.63 94 84.5 1829(SEQ ID NO:19) H-HGDGSFSSELSTILDNLAARDFIAWLIQTKKKKKKK-NH2 4072.26 4072.595 51.8 1830 (SEQ ID NO:20) H-HGDGSFSDELSTILDNLAARDFIAWLIQTK-NH2 3331.683331.88 94 26.4 1831 (SEQ ID NO:21) H-HGDGSFTDELSTILDNLAARDFIAWLIQTK-NH23345.7 3345.38 94 46.0 1832 (SEQ ID NO:22)H-HGDGSFTSELSTILDNLAARDFIAWLIQTK-NH2 3317.7 3117.88 94 17.5 1833 (SEQ IDNO:23) H-HGDGSFSSELSTILDNLAARDFIAWLIQTK-NH2 3303.69 3304.13 97 34.5 1834(SEQ ID NO:24) H-HGDGSFTSELATILDNLAARDFIAWLIQTK-NH2 3301.71 3301.75 9416.0 1835 (SEQ ID NO:25) H-HGDGSFSDELKTILDNLAARDFIAWLIQTK-NH2 3372.753373.13 94 89.7 1836 (SEQ ID NO:26) H-HGDGSFTDELKTILDNIAARDFIAWLIQTK-NH23386.76 3386.88 92 26.0 1839 (SEQ ID NO:27)H-HGDGSFSDELATILDNLAARDFIAWLIQTKITDKKKKKK-NH2 4413.42 4413.88 92 18.21840 (SEQ ID NO:28) H-HGDGSFSDELATILDNLAARDFIAWLIQTKITD-NH2 3644.853644.88 95 21.3 1841 (SEQ ID NO:29) H-HGDGSFSDELATILDNLAARDFIAWLIQTK-NH23315.69 3315.88 91 73.6 1842 (SEQ ID NO:30)H-HGDGSFTSELKTILDNLAARDFIAWLIQTK-NH2 3358.77 3358.88 97 26.3 1843 (SEQID NO:31) H-HGDGSFTDELATILDNLAARDFIAWLIQTK-NH2 3329.7 3329.88 90 23.61844 (SEQ ID NO:32) H-HGEGTFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH24329.42 4329.63 90 46.0 1845 (SEQ ID NO:33)H-HGEGTFSDELSTILDALAARDFIAWLIATKITDKKKKKK-NH2 4357.42 4357.38 93 52.51846 (SEQ ID NO:34) H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH24315.41 4315.38 90 28.8 1847 (SEQ ID NO:35)H-HGEGSFSDELSTILDALAARDFIAWLIATKITDKKKKKK-NH2 4343.4 4343.5 90 59.4 1848(SEQ ID NO:36) H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 4313.434313.63 90 230.0 1849 (SEQ ID NO:37)H-HGEGSFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 4299.41 4299.5 97 68.01850 (SEQ ID NO:38) H-HGEGSFSDELKTILDALAARDFIAWLIATKITDKKKKKK-NH24384.46 4384.63 93 38.0 1851 (SEQ ID NO:39)H-HGEGTFSDELKTILDALAARDFIAWLIATKITDKKKKKK-NH2 4398.48 4398.38 95 90.61852 (SEQ ID NO:40) H-HGEGTFSSELKTILDALAARDFIAWLIATKITDKKKKKK-NH24370.48 4370.63 95 63.2 1853 (SEQ ID NO:41)H-HGEGTFSSELSTILDALAARDFIAWLIATKITD-NH2 3560.85 3560.13 97 18.0 1854(SEQ ID NO:42) H-HGEGTFSDELSTILDALAARDFIAWLIATKITD-NH2 3588.85 3589.1396 12.5 1855 (SEQ ID NO:43) H-HGEGSFSSELSTILDALAARDFIAWLIATKITD-NH23546.84 3547 96 27.3 1856 (SEQ ID NO:44)H-HGEGSFSDELSTILDALAARDFIAWLIATKITD-NH2 3574.83 3575 96 18.0 1857 (SEQID NO:45) H-HGEGTFSSELATILDALAARDFIAWLIATKITD-NH2 3544.86 3544.88 9439.3 1858 (SEQ ID NO:46) H-HGEGSFSSELATILDALAARDFIAWLIATKITD-NH2 3530.843530.88 90 43.3 1859 (SEQ ID NO:47)H-HGEGSFSDELKTILDALAARDFIAWLIATKITD-NH2 3615.89 3615.13 90 11.4 1860(SEQ ID NO:48) H-HGEGTFSDELKTILDALAARDFIAWLIATKITD-NH2 3629.91 3629.1392 13.9 1861 (SEQ ID NO:49) H-HGEGTFSSELKTILDALAARDFIAWLIATKITD-NH23601.91 3601.13 99 16.2 *Yield; yield/g resin

Example 1 Synthesis of Compound 1846 (SEQ ID NO:34)

H-His-Gly-Glu-Gly-Ser-Phe-Ser-Ser-Glu-Leu-Ser-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys-Lys-Lys-Lys-NH₂(SEQ ID NO:34) on TentaGel S RAM-Lys(Boc)Fmoc.

Dry TentaGel S RAM-Lys(Boc)Fmoc (0.24 mmol/g, 1 g) was placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand treated as described under “batchwise peptide synthesis on TentaGelresin” until finishing the coupling of the N-terminal Histidine. Allcouplings were continued over night. The acylations were checked asearlier described. After completed synthesis and deprotection of theN-terminal Fmoc group the peptide was cleaved from the resin asdescribed above. After purification using preparative HPLC as earlierdescribed, 28.8 mg peptide product was collected with a purity betterthan 90% and the identity of the peptide was confirmed by MS (found M4315.38, calculated M 4315.41).

Example 2 Synthesis of Compound 1848 (SEQ ID NO:36)

H-His-Gly-Glu-Gly-Thr-Phe-Ser-Ser-Glu-Leu-Ala-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys-Lys-Lys-Lys-NH₂(SEQ ID NO:36) on TentaGel S RAM-Lys(Boc)Fmoc.

Dry TentaGel S RAM-Lys(Boc)Fmoc (0.24 mmol/g, 1 g) was placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand treated as described under “batchwise peptide synthesis on TentaGelresin” until finishing the coupling of the N-terminal Histidine. Allcouplings were continued over night. The acylations were checked asearlier described. After completed synthesis and deprotection of theN-terminal Fmoc group the peptide was cleaved from the resin asdescribed above. After purification using preparative HPLC as earlierdescribed, 230 mg peptide product was collected with a purity betterthan 90% and the identity of the peptide was confirmed by MS (found M4313.63, calculated M 4313.43).

Example 3 Synthesis of Compound 1855 (SEQ ID NO:43)

H-His-Gly-Glu-Gly-Ser-Phe-Ser-Ser-Glu-Leu-Ser-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-NH₂(SEQ ID NO:43) on TentaGel S RAM-Asp(OtBu)Fmoc.

Dry TentaGel S RAM-Asp(OtBu)Fmoc (0.2 mmol/g, 1 g) was placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand treated as described under “batchwise peptide synthesis on TentaGelresin” until finishing the coupling of the N-terminal Histidine. Allcouplings were continued over night. The acylations were checked asearlier described. After completed synthesis and deprotection of theN-terminal Fmoc group the peptide was cleaved from the resin asdescribed above. After purification using preparative HPLC as earlierdescribed, 27.3 mg peptide product was collected with a purity betterthan 96% and the identity of the peptide was confirmed by MS (found M3547, calculated M 3546.84).

Example 4 Synthesis of Compound 1857 (SEQ ID NO:45)

H-His-Gly-Glu-Gly-Thr-Phe-Ser-Ser-Glu-Leu-Ala-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-NH₂(SEQ ID NO:45) on TentaGel S RAM-Asp(OtBu)Fmoc.

Dry TentaGel S RAM-Asp(OtBu)Fmoc (0.2 mmol/g, 1 g) was placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand treated as described under “batchwise peptide synthesis on TentaGelresin” until finishing the coupling of the N-terminal Histidine. Allcouplings were continued over night. The acylations were checked asearlier described. After completed synthesis and deprotection of theN-terminal Fmoc group the peptide was cleaved from the resin asdescribed above. After purification using preparative HPLC as earlierdescribed, 39.3 mg peptide product was collected with a purity betterthan 94% and the identity of the peptide was confirmed by MS (found M3544.88, calculated M 3544.86).

Example 5 Synthesis of Compound 1846 A (SEQ ID NO:34) (Acetate Salt)

Counter ion exchange from trifluoroacetate to acetate of Compound 1846(SEQ ID NO:34). The purified synthetic peptide product of compound 1846(SEQ ID NO:34) is isolated as a trifluoroacetate salt, due to thepresence of trifluoroacetic acid (0.1% v/v) in the HPLC buffers used forthe purification of the crude synthetic peptide product.

In order to exchange the counter ion trifluoroacetate with acetate, asolution of the peptide was passed through a column packed with strongbase ion exchange resin on the acetate (Dowex 1×8). 365 mg Compound 1 isdissolved in 40 ml water. The solution is passed through a columncontaining 40 ml strong base ion exchange resin on the acetate (Dowex1×8; capacity 1.33 meq/ml resin). The resin is then washed with 4×30 mlwater and the eluate is collected and lyophilized resulting in 312 mgacetate salt with a purity according to HPLC analysis of 97%.

Example 6 Synthesis of Compound 1848 (SEQ ID No:36) C (Chloride Salt)

Counter ion exchange from trifluoroacetate (Tfa) to chloride (Cl—) ofCompound 1848 (SEQ ID NO:36).

100 mg Compound 1 was dissolved in 50 ml 0.1M hydrochloric acid and theresulting solution was lyophilized. The remanence was dissolved in 50 mlwater and lyophilized again resulting in 80 mg of the chloride salt witha purity according to HPLC of 93%.

Example 7 Chemical Stability Testing

The GLP-2 analogues were dissolved in purified water and subsequentlydiluted in solutions containing HCl, NaOH, H₂O₂ or NH₄HCO₃. Thesolutions were incubated at 40° C. to produce hydrolysis, deamidationand oxidation products. The samples were analyzed by RP-HPLC and thepercentage of remaining intact compound was determined as a measure forthe relative stability. The major degradation products were tentativelyidentified by LC-MS. The peptides used are listed in Table 3.

TABLE 2 GLP-2 analogues tested and compared for chemical stability.Peptide Purity Compound Batch Monoisotopic Content RP-HPLC No. No. MW(g/mol) (%) (%) ZP1559 70.30 3749.80 90 96 (SEQ ID NO:54) AB and K 1AZP1820 78.65 4070.28 80 94 (SEQ ID NO:10) 1A ZP1834 78.90 3301.71 88 94(SEQ ID NO:24) 1A ZP1846 107.07 4315.41 80 90 (SEQ ID NO:34) 1A ZP184891.58 4313.43 80 90 (SEQ ID NO:36) 1A ZP1849 91.60 4299.41 79 97 (SEQ IDNO:37) 1A ZP1855 88.49 3546.84 89 96 (SEQ ID NO:43) 1A-2- 1A ZP1857108.01 3544.86 89 94 (SEQ ID NO:45) X-1A ZP1858 108.05 3530.84 89 90(SEQ ID NO:46) 1A

Compound ZP1559 (SEQ ID NO:54), Gly²-GLP-2, was used as a reference forthe other GLP-2 analogues tested. In the GLP-2 analogues, positionswhere the sequence differs from the reference compound ZP1559 (SEQ IDNO:54) are shown in bold font. The sequences are listed in Table 3.

The compounds are listed as pairs according to their sequences and withand without C-terminal -K₆ extension.

TABLE 3 Sequences of Gly²-GLP-2 and GLP-2 analogues. Compound SequenceZP1559 HGDGS FSDEM NTILD NLAAR DFINW LIQTK (SEQ ID NO:54) ITD-OH ZP1820HGDGS FTSEL ATILD NLAAR DFIAW LIQTK (SEQ ID NO:10) K ₆-NH₂ ZP1834 HGDGSFTSEL ATILD NLAAR DFIAW LIQTK (SEQ ID NO:24) -NH ₂ ZP1846 HGEGS FSSELSTILD ALAAR DFIAW LIATK (SEQ ID NO:34) ITD K ₆-NH₂ ZP1855 HGEGS FSSELSTILD ALAAR DFIAW LIATK (SEQ ID NO:43) ITD-NH ₂ ZP1848 HGEGT FSSEL ATILDALAAR DFIAW LIATK (SEQ ID NO:36) ITD K ₆-NH₂ ZP1857 HGEGT FSSEL ATILDALAAR DFIAW LIATK (SEQ ID NO:45) ITD-NH ₂ ZP1849 HGEGS FSSEL ATILD ALAARDFIAW LIATK (SEQ ID NO:37) ITD K ₆-NH₂ ZP1858 HGEGS FSSEL ATILD ALAARDFIAW LIATK (SEQ ID NO:46) ITD-NH ₂

Chemicals used in the experiment are listed in Table 4.

TABLE 4 Chemicals and reagents used for the analytical procedures.Substance Quality Supplier Product Acetonitrile (MeCN) HPLC gradeRiedel-deHaen 34851 Trifluoroacetic 99.9% Pierce 28904 Acid (TFA) FormicAcid (FA) 98-100% Merck 1.00264.1000 Hydrochloric acid HCl “Baker J. T.Baker  7088 Analyzed” Sodium Hydroxide “Baker J. T. Baker  7098 NaOHAnalyzed” Hydrogen Peroxide H₂O₂ 30% w/w Sigma-Aldrich H1009 NH₄HCO₃99.0% AnalaR 103025E

The water was first de-mineralized to a resistance of ≧18 MΩcm and thenpassed through a Milli-Q system (Millipore, Bedford, USA). The Milli-Qpurified water (MQW) was finally tapped through a 0.22-μm sterile filter(Millipak 40 Gamma Gold, Millipore).

The GLP-2 analogues tested and the reference Gly2-GLP-2, ZP1559 (SEQ IDNO:54), were dissolved in water and subsequently diluted into solutionscontaining HCl, NaOH, H₂O₂ or NH₄HCO₃.

The samples were incubated at 40° C. to generate hydrolysis, deamidationand oxidation products, respectively. The compounds were analysed byRP-HPLC for purity of the original main peak and by LC-MS forconfirmation of the identity by mass of the main peak and majordegradation products.

Preparation of Stress Solutions:

0.2 M HCl: 4 mL MQW and 1 mL of 1 M HCl. 0.02 M NaOH: 4 mL MQW and 1 mLof 0.1 M NaOH. 0.2 M NH₄HCO₃, pH 8: 0.79 g NH₄HCO₃ was dissolved in 50mL MQW. 1% H₂O₂: 5.8 mL MQW and 0.2 mL of 30% H₂O₂.Sample Solutions:

The GLP-2 analogues were first dissolved in MQW to a concentration of 4mg/mL and then further diluted in the stress solutions at a 1:1 ratio(e.g. 125 μL plus 125 μL). The final concentrations were 2 mg/mL of theGLP-2 analogues for stress conditions with 0.1 M HCl, 0.01 M NaOH, 0.1 MNH₄HCO₃ and 0.5% H₂O₂, respectively.

The solutions were incubated at 40° C. in the dark and then diluted inEluent A to a concentration of 0.5 mg/mL (addition of 750 μL) prior toanalyses by RP-HPLC and by LC-MS.

TABLE 5 Conditions for stress test. Solution 0.1 M 0.5% 0.1 M HCl 0.01MNaOH NH₄HCO₃ H₂O₂ Temperature (° C.) 40 40 40 40 Storage (days) 12 3 6 3RP-HPLC

The RP-HPLC analyses were performed on an Agilent Series 1100 HPLCsystem under control of the ChemStation (Revision A.08.03 [847])software from Agilent Technologies, Inc. The raw data and the results ofthe peak integration were deposited on the ChemStore C/S server by theuse of Agilent Technologies Revision B01.03 software.

TABLE 6 The RP-HPLC method. Method file name P2204071.M Column Vydac218MS52, 5 μm, 300 Å, 2.1 × 250 mm Gradient (time; % B) 0; 5, 2; 5, 7;15, 25; 30, 45; 40, 65; 50, 70; 100, 73; 100, 75; 5, 90; 5 Eluent A0.05% TFA, 0.05% FA in MQW Eluent B 0.05% TFA, 0.05% FA in MeCN FlowRate 0.200 mL/min Injection Volume 20 μL Column Temperature 25° C. AutoSampler Temp. 4° C. UV detection 220 nmLC-MS

Analytical LC-MS analyses were performed on an Agilent Technologies 1100HPLC instrument consisting of an on-line degasser, quaternary gradientpump, an auto sampler, a column oven, an UV detector. The HPLCinstrument was interfaced with a Micromass LCT (ESI-TOF) massspectrometer under control of Masslynx 3.5 software, from MicroMass, UK.

TABLE 7 The LC-MS method Method file name P22_04_071_003.M Column Vydac218MS52, 5 μm, 300 Å, 2.1 × 250 mm Gradient (time; % B) 0; 5, 2; 5, 7;15, 25; 30, 45; 40, 65; 50, 70; 100, 73; 100, 75; 5, 90; 5 Eluent A0.05% TFA, 0.05% FA in MQW Eluent B 0.05% TFA, 0.05% FA in MeCN FlowRate 0.200 mL/min Injection Volume 30 μL Column Temperature 25° C. AutoSampler Temp. 4° C. UV detection 220 nm

TABLE 8 The MS set-up according to SOP 22-3003. Cone voltage 30 VCapillary voltage 3.1 kV Nitrogen nebuliser gas flow 100 L/hrDesolvation gas flow 500 L/hr Desolvation temperature 250° C. Sourceblock temperature 100° C.

The results are shown in Table 9 as the compound purity measured byRP-HPLC after incubation under stress conditions. This purity is ameasure for the remaining intact compound after incubation in stresssolutions, relative to the purity measured a T=0. These results do nottake into account possible hidden degradation products not observed bythis analytical RP-HPLC method.

Major degradation products in the stress test samples were tentativelyidentified by the LC-MS method. Any isomers to the parent compounds andminor degradation products were not observed by this analytical LC-MSmethod. Tentative identifications are listed in Tables 11 to 15.

The GLP-2 analogues tested are listed as pairs according to theirssequences with and without the C-terminal -K₆ extension.

TABLE 9 Observed purity of test compounds after incubation under stressconditions. Purity Purity Sequence Purity H₂O₂ NH₄HCO₃ +/− HCl 0.1 M 0.5% 0.1 M GLP-2 C-terminal 12 days 3 days 6 days analogues K₆ (%) (%) (%)ZP1559 Reference 57 <5 66 (SEQ ID NO:54) ZP1820 K₆ 45 62 85 (SEQ IDNO:10) ZP1834 — 38 53 91 (SEQ ID NO:24) ZP1846 K₆ 70 64 82 (SEQ IDNO:34) ZP1855 — 85 14 97 (SEQ ID NO:43) ZP1848 K₆ 63 68 93 (SEQ IDNO:36) ZP1857 — 86 59 96 (SEQ ID NO:45) ZP1849 K₆ 64 78 86 (SEQ IDNO:37) ZP1858 — 88 60 91 (SEQ ID NO:46)

The results for the compounds incubated in NaOH are not listed, becauseno difference in the stability could be observed. The degradationproducts and the main peak all have the same mass by LC-MS analysis;these compounds were probably racemized over time. The results in Table9 show that the GLP-2 analogues tested in general are more chemicallystable than the Gly2-GLP-2 reference, ZP1559 (SEQ ID NO:54).

During acid hydrolysis the GLP-2 analogues tested are more stable thanthe Gly²-GLP-2 reference, ZP1559 (SEQ ID NO:54), except for the ZP1820(SEQ ID NO:10) and ZP1834 (SEQ ID NO:24). This is mainly due to theamino acid Asp in position 3. Glu rather than Asp may minimize thecleavage between amino acid 3 and 4 Asp-Gly. The other ZP GLP-2analogues tested do have approximately the same stability and with atendency of slightly higher stability for the compounds withoutC-terminal -K₆, ZP1855 (SEQ ID NO:43), ZP1857 (SEQ ID NO:45), and ZP1858(SEQ ID NO:46). This difference is explained by the amino acid inposition 33 Asp. In the compound without C-terminal -K₆, this amino acidis C-terminal and a cleavage between amino acid 32 and 33 Tyr-Asp occursslower than for a cleavage between amino acid 33 and 34 Asp-Lys. Thedifference in the stability is due to the Asp and not to the C-terminal-K₆.

Under the conditions of accelerated oxidation (H₂O₂, se also Table 10and 13), the GLP-2 analogues tested are much more stable than theGly²-GLP-2 reference, ZP1559 (SEQ ID NO:54). This is probably due tooxidation of Met in position 10 in ZP1559 (SEQ ID NO:54). An exceptionis ZP1855 (SEQ ID NO:47), which shows an unexplainable low stability.This could be specific for the batch of ZP1855 (SEQ ID NO:47) andfurther studies will be needed to explain this.

The stability under conditions promoting deamidation (NH₄HCO₃), theGLP-2 analogues tested are more stable than the Gly2-GLP-2 reference,ZP1559 (SEQ ID NO:54). This is probably due to several deamidation sitesin ZP1559 (SEQ ID NO:54), Asn in position 11, 16 and 24, which are notpresent in the sequences for the GLP-2 analogues tested.

Tentative Identification of Major Degradation Products by LC-MS

TABLE 10 Major cleavage sites in ZP GLP-2 analogues and the referenceZP1559 (SEQ ID NO:54). ZP1559HGD^(↓)GS FSDEM N^(↓)TILD NLAAR (SEQ IDNO:54) DFINW LIQTK ITD-OH ZP1820HGD^(↓)GS FTSEL ATILD NLAAR (SEQ IDNO:10) DFIAW LIQTK K₆-NH₂ ZP1834HGD^(↓)GS FTSEL ATILD NLAAR (SEQ IDNO:24) DFIAW LIQTK -NH₂ ZP1846HGEGS FSSEL STILD ALAAR DFIAW (SEQ IDNO:34) LIATK ITD^(↓)K₆-NH₂ ZP1855HGEGS FSSEL STILD ALAAR DFIAW (SEQ IDNO:43) LIATK IT^(↓)D-NH2 ZP1848HGEGT FSSEL ATILD ALAAR DFIAW (SEQ IDNO:36) LIATK ITD^(↓)K₆-NH₂ ZP1857HGEGT FSSEL ATILD ALAAR DFIAW (SEQ IDNO:45) LIATK IT^(↓)D-NH₂ ZP1849HGEGS FSSEL ATILD ALAAR DFIAW (SEQ IDNO:37) LIATK ITD^(↓)K₆-NH₂ ZP1858HGEGS FSSEL ATILD ALAAR DFIAW (SEQ IDNO:46) LIATK IT^(↓)D-NH₂Solutions Stressed by HCl

TABLE 11 GLP-2 analogues incubated 12 days at 40° C. in 0.1 M HCl. GLP-2Measured Theoretical Difference Major/minor abundance analogues MW (Da)MW (Da) Mass (Da) Possible ID suggestion ZP1559 3749.88 3749.80 +0.08Major product (SEQ ID NO:54) 3732.75 Na −17.13 Minor, cyclic deamidation3751.88 Na +2.00 Minor, 2 × deamidation ZP1820 4070.13 4070.28 −0.15Major product A¹-A³⁶ (SEQ ID NO:10) 4071.25 Na +1.12 Minor, deamidation3761.25 3761.17 +0.08 Minor, hydrolysis A⁴-A³⁶ 2526.75 2526.56 +0.19Minor, hydrolysis A¹⁶-A³⁶ 3762.25 3762.16 +0.09 Minor, hydro. A⁴-A³⁶,dea. ZP1834 3302.00 3301.71 +0.29 Major product (SEQ ID NO:24) 3303.00Na +1.00 Minor, deamidation 3283.88 Na −18.12 Minor, cyclic deamidation3302.88 Na +0.88 Minor, deamidation 2992.88 2992.60 −309.12 Major,hydro. A⁴-A³⁰, dea. 2993.88 2993.59 −308.12 Minor, hydrolysis A⁴-A³⁰2993.75 2993.59 −308.25 Minor, hydrolysis A⁴-A³⁰ ZP1846 4315.00 4315.41+0.59 Major product A¹-A³⁹ (SEQ ID NO:34) 3547.50 3547.82 −0.32 Minor,hydrolysis A¹-A³³ 3934.88 3935.26 −0.38 Minor, hydrolysis A⁵-A³³ 2755.382755.70 −0.32 Minor, hydrolysis A¹⁶-A³⁹ 2158.25 2158.37 −0.12 Minor,hydrolysis A²²-A³⁹ 3155.16 Na −1160.25 No suggestion ZP1855 3548.133546.84 +1.29 Major product/deamida. (SEQ ID NO:43) 3548.13 Na +1.29Minor, deamidation 3225.13 3223.71 +1.42 Minor, hydro. A⁴-A³³, dea.3433.13 3432.79 +0.34 Minor, hydrolysis A¹-A³² 3354.50 3352.76 +1.76Minor, hydro. A³-A³³, dea. ZP1857 3544.50 3544.86 −0.36 Major productA¹-A³³ (SEQ ID NO:45) 3430.63 3430.81 −0.18 Minor, hydrolysis A¹-A³²3351.38 3350.78 +0.60 Minor, hydrolysis A³-A³³ 3165.50 3164.71 +0.79Minor, hydrolysis A⁵-A³³ 3222.50 3221.73 +0.77 Minor, hydrolysis A⁴-A³³2830.25 2829.56 +0.69 Minor, hydrolysis A⁸-A³³ 3545.13 Na +0.63 Minor,recemization ZP1849 4299.63 4299.41 +0.22 Major product, A¹-A³⁹ (SEQ IDNO:37) 2755.88 2755.70 +0.18 Minor, hydrolysis, A¹⁶-A³⁸ 3532.00 3531.82+0.18 Minor, hydrolysis, A¹-A³³ 2158.38 2159.05 −0.67 Minor, hydrolysis,2158.37 +0.01 A¹-A²¹ or A²²-A³⁹ 3976.88 3976.29 +0.59 Minor, hydrolysis,A⁴-A³⁹ 4105.13 4105.33 −0.20 Minor, hydrolysis, A³-A³⁹ 3920.50 3919.27+1.23 Minor, hydro., A⁵-A³⁹, dea. 1561.84 1561.73 +0.11 Minor,hydrolysis, A¹-A¹⁵ ZP1858 3532.13 3530.84 +1.29 Major product/deamida.(SEQ ID NO:46) Na = not available, no theoretical MW was calculated orsuggested. *Mass difference is the measured MW from the theoretical MWfor the main peak or for the suggested compound.

The results in Table 12 show that the measured molecular weight for theGLP-2 analogues tested and the Gly²-GLP-2 reference, ZP1559 (SEQ IDNO:54), correspond to the theoretical molecular weight.

The most abundant degradation products are the cleavage between aminoacids 3 and 4; Asp and Gly in ZP1559 (SEQ ID NO:54), ZP1820 (SEQ IDNO:10), and ZP1834 (SEQ ID NO:24). For the compounds ZP1846 (SEQ IDNO:34), ZP1848 (SEQ ID NO:36), and ZP1849 (SEQ ID NO:37), which are withC-terminal -K₆, degradation products corresponding to a loss ofC-terminal -K₆ (Lys₆) in position 33-39 were detected. For the compoundsZP1855 (SEQ ID NO:43) and ZP1857 (SEQ ID NO:45), which are withoutC-terminal -K₆, degradation products corresponding to a loss ofC-terminal Asp in position 33 were detected.

Minor degradation products detected were cleavage between amino acids 15and 16 (Asp and Asn or Asp and Ala), amino acids 4 and 5 (Gly and Ser),amino acids 21 and 22 (Asp and Phe).

Solutions Stressed by H₂O₂

TABLE 12 GLP-2 analogues incubated 3 days at 40° C. in 0.5 % H₂O₂. GLP-2Measured Theoretical Difference* Major/minor abundance analogues MW (Da)MW (Da) Mass (Da) Possible ID suggestion ZP1559 n.d. 3749.80 — Noproduct intact (SEQ ID NO:54) 3766.00 Na +16.2 Major, oxidation of MZP1820 4070.63 4070.28 −0.35 Major product (SEQ ID NO:10) 4052.38 Na−18.25 Major, deamidation precursor 4086.50 Na +15.87 Minor, oxidationof W 4068.75 Na −1.88 Minor, 2 × deamidation ZP1834 3301.75 3301.71+0.04 Major product (SEQ ID NO:24) 3283.75 Na −18.00 Major, deamidationprecursor 3317.88 Na +16.13 Minor, oxidation of W 3299.75 Na −2.00Minor, 2 × deamidation ZP1846 4315.50 4315.41 +0.09 Major product (SEQID NO:34) 4331.88 Na +16.38 Minor, oxidation of W ZP1855 3547.00 3546.84+0.16 Major product (SEQ ID NO:43) 3481.88 Na −65.12 Minor, no suggest3410.00 Na −137.00 Minor, A²-A³³ 3563.00 Na +16.00 Minor, oxidation of WZP1848 4313.50 4313.43 +0.07 Major product (SEQ ID NO:36) 4329.63 Na+16.13 Minor, oxidation of W ZP1857 3545.00 3544.86 +0.14 Major product(SEQ ID NO:45) 3408.00 Na −137.00 Minor, A²-A³³ 3561.00 Na +16.00 Minor,oxidation of W 3479.88 Na −65.12 Minor, no suggest ZP1849 4299.504299.41 +0.09 Major product (SEQ ID NO:37) 4315.75 Na +16.25 Minor,oxidation of W ZP1858 3531.00 3530.84 +0.16 Major product (SEQ ID NO:46)3394.13 Na −136.87 Minor, A²-A³³ 3547.00 Na +16.00 Minor, oxidation of W3466.00 Na −65.00 Minor, no suggest Na = not available, no theoreticalMW was calculated or suggested. *Mass difference is the measured MW fromthe theoretical MW for the main peak.

The results in Table 13 show that the measured molecular weight for theGLP-2 analogues tested and the Gly²-GLP-2 reference, ZP1559 (SEQ IDNO:54), correspond to the theoretical molecular weight. For ZP1559 (SEQID NO:54), a major degradation product with a MW of +16 Da is observed,which could probably be the oxidized products of Met in position 10. ForZP1820 (SEQ ID NO:10) and ZP1834 (SEQ ID NO:24), a major degradationproduct with a MW of +18 Da is observed. They could probably be loss ofwater in the precursor to a deamidation. In addition, minor productswith MW of −2 Da is observed and could be deamidation in two sites.Minor products with MW of +16 Da is observed in all compounds and couldprobably be oxidation of Trp. For the compounds ZP1855 (SEQ ID NO:43),ZP1857 (SEQ ID NO:45), and ZP1858 (SEQ ID NO:46), which all are withoutC-terminal -K₆, minor degradation products with MW of −137 Da and of −65Da are detected, corresponding to a loss of His in position 1 and anunidentified degradation product, respectively.

Solutions Stressed by NaOH

TABLE 13 GLP-2 analogues incubated 3 days at 40° C. in 0.01 M NaOH.GLP-2 Measured Theoretical Difference* analogues MW (Da) MW (Da) Mass(Da) Comments ZP1559 3750.35 3749.80 +0.58 No change in (SEQ ID NO:54)MW ZP1820 4070.63 4070.28 +0.35 No change in (SEQ ID NO:10) MW ZP18343302.00 3301.71 +0.29 No change in (SEQ ID NO:24) MW ZP1846 4315.634315.41 +0.22 No change in (SEQ ID NO:34) MW ZP1855 3547.25 3546.84+0.41 No change in (SEQ ID NO:43) MW ZP1848 4313.88 4313.43 +0.45 Nochange in (SEQ ID NO:36) MW ZP1857 3545.13 3544.86 +0.27 No change in(SEQ ID NO:45) MW ZP1849 4299.88 4299.41 +0.47 No change in (SEQ IDNO:37) MW ZP1858 3531.13 3530.84 +0.29 No change in (SEQ ID NO:46) MW na= not available, no theoretical MW was calculated or suggested. *Massdifference is the measured MW from the theoretical MW for the main peak.

The LC-MS analyses show the same molecular weight in all peaks for eachcompound. This means that the most abundant degradation products areprobably racemization from L- to D-form of one or more of the aminoacids in the sequence. The main peak can then hide one or severalracemized products, thus no residual purity of intact peptide could bedetermined. No major degradation products from cleavage have beendetected.

Solutions Stressed by NH₄HCO₃

TABLE 14 GLP-2 analogues incubated 6 days at 40° C. in 0.1 M NH₄HCO₃, pH8 GLP-2 Measured Theoretical Difference* Major/minor abundance analoguesMW (Da) MW (Da) Mass (Da) Possible ID suggestion ZP1559 3750.00 3749.80+0.20 Major product (SEQ ID NO:54) 3751.13 Na +1.13 Minor, possibledeamidation 3751.13 Na +1.13 Minor, Possible deamidation ZP1820 4070.134070.28 −0.15 Major product (SEQ ID NO:10) 4070.13 Na −0.15 Minor,possible racemization ZP1834 3302.00 3301.71 +0.29 Major product (SEQ IDNO:24) 3302.00 Na +0.29 Minor, possible racemization ZP1846 4315.254315.41 −0.16 Major product (SEQ ID NO:34) 4315.63 Na +0.38 Minor,possible racemization ZP1855 3547.13 3546.84 +0.29 Major product (SEQ IDNO:43) ZP1848 4313.75 4313.43 +0.32 Major product (SEQ ID NO:36) ZP18573544.75 3544.86 −0.11 Major product (SEQ ID NO:45) ZP1849 4299.504299.41 +0.09 Major product (SEQ ID NO:37) ZP1858 3531.00 3530.84 +0.16Major product (SEQ ID NO:46) Na = not available, no theoretical MW wascalculated or suggested. *Mass difference is the measured MW from thetheoretical MW for the main peak.

The results in Table 14 show that the measured molecular weight for theGLP-2 analogues tested and the Gly2-GLP-2 reference, ZP1559 (SEQ IDNO:54), correspond to the theoretical MW. For ZP1559, minor degradationproducts with a MW of +1 Da are observed, which could probably bedeamidated products. For ZP1820 (SEQ ID NO:10), ZP1834 (SEQ ID NO:24),and ZP1846 (SEQ ID NO:34), minor degradation products with the same MWas for the main compound are observed. They could probably be racemizedproducts or deamidation. However, the MS resolution of the presentinstrument was not adequate to confirm or reject this. In addition,these products could be present in the other compounds, but not detectedas they could be hidden under the main peak.

All the present GLP-2 analogues tested have better chemical stabilitycompared with the reference compound Gly2-GLP-2; 1559 (SEQ ID NO:54),under stress conditions for hydrolysis, oxidation and deamidation. Thecompounds 1820 (SEQ ID NO:10) and 1834 (SEQ ID NO:24) are less stablethan the remaining six candidates due to acid hydrolysis. Highestchemical stability was seen when amino acid A³ was Glu rather than Asp.No significant difference in the chemical stability of the remaining sixcandidates were observed, except a slightly better stability without-K₆, which were mainly due to a labile site after Asp and the loss of-K₆ in candidates having the C-terminal -K₆.

Example 8 Screening for Intestinal Growth Effects of Compounds in C57BLMice

The ability of the present compounds to stimulate intestinal growth wasdetermined in male C57BL mice. Individual groups (n=6) of mice weregiven 30 nmol/kg of each compound, s.c, twice daily for ten consecutivedays. For comparison purposes other groups of animals were given eitheran equimolar dose of [Gly2]GLP-2 (SEQ ID NO:54) or vehicle (phosphatebuffered saline, pH 7.4) in the same dosing regimen. Twenty-four hoursafter the last dose of compound had been given the mice were sacrificedand the small intestine (from the pylorus to the cecum) and the colon(intestine distal to cecum) was emptied and weighed. To correct forslight difference in body weight (BW), the organ mass of the smallintestine (SI) and colon were expressed relative to BW. Thenon-selective reference compound [Gly2]GLP-2 (SEQ ID NO:54) has beenreported to stimulate gastrointestinal growth in both esophagus,stomach, small intestine and colon and to evaluate differences in growthpattern induced by compounds, the small intestine-colon sensitivityindex of compound X was calculated as:(SI/Colon)_(x)/(SI/Colon)_([Gly2]GLP-2)%

Compounds with a small intestine-colon sensitivity greater than or equalto 1.05 were considered relatively selective for the small intestine(Table 15).

Compounds with a small intestine-colon sensitivity smaller than or equalto 0.95 were considered relatively selective for the colon (Table 15).

TABLE 15 List of selective GLP-2 analogue compounds. Position 1 2 3 4 56 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3031 32 33 % [Gly2]GLP-2 Non-selective reference compound = [Gly2]GLP-2(SEQ ID NO:54) H G D G S F S D E M N T I L D N L A A R D F I N W L I Q TK I T D OH 100 100 100 Small intestine selective compounds (SEQ IDNOS:32-34, 36-41, 43, 45-57) 1844 H G E G T F S S E L S T I L D A L A AR D F I A W L I A T K I T D K6 NH2 106 89 119 1845 H G E G T F S D E L ST I L D A L A A R D F I A W L I A T K I T D K6 NH2 105 98 107 1846 H G EG S F S S E L S T I L D A L A A R D F I A W L I A T K I T D K6 NH2 120102 118 1848 H G E G T F S S E L A T I L D A L A A R D F I A W L I A T KI T D K6 NH2 109 93 117 1849 H G E G S F S S E L A T I L D A L A A R D FI A W L I A T K I T D K6 NH2 109 95 115 1850 H G E G S F S D E L K T I LD A L A A R D F I A W L I A T K I T D K6 NH2 103 89 116 1851 H G E G T FS D E L K T I L D A L A A R D F I A W L I A T K I T D K6 NH2 109 96 1141852 H G E G T F S S E L K T I L D A L A A R D F I A W L I A T K I T DK6 NH2 106 97 109 1853 H G E G T F S S E L S T I L D A L A A R D F I A WL I A T K I T D NH2 101 96 105 1855 H G E G S F S S E L S T I L D A L AA R D F I A W L I A T K I T D NH2 106 88 120 1857 H G E G T F S S E L AT I L D A L A A R D F I A W L I A T K I T D NH2 117 90 130 1858 H G E GS F S S E L A T I L D A L A A R D F I A W L I A T K I T D NH2 104 96 1081859 H G E G S F S D E L K T I L D A L A A R D F I A W L I A T K I T DNH2 83 73 114 Colon selective compounds (SEQ ID NOS:20, 21, 25-29, 31)1830 H G D G S F S D E L S T I L D N L A A R D F I A W L I Q T K NH2 100106 94 1831 H G D G S F T D E L S T I L D N L A A R D F I A W L I Q T KNH2 90 102 88 1835 H G D G S F S D E L K T I L D N L A A R D F I A W L IQ T K NH2 96 105 91 1836 H G D G S F T D E L K T I L D N L A A R D F I AW L I Q T K NH2 94 105 90 1839 H G D G S F S D E L A T I L D N L A A R DF I A W L I Q T K I T D K6 NH2 112 133 84 1840 H G D G S F S D E L A T IL D N L A A R D F I A W L I Q T K I T D NH2 113 131 86 1841 H G D G S FS D E L A T I L D N L A A R D F I A W L I Q T K NH2 113 126 90 1843 H GD G S F T D E L A T I L D N L A A R D F I A W L I Q T K NH2 111 130 85Results

The intestinal growth effects of the present compounds according to theinvention were determined based on the ability of the peptides todose-dependently increase SI mass relative to the effect of equimolardoses of the non-selective reference compound [Gly₂]GLP-2 (SEQ IDNO:54).

The findings from this study demonstrate that GLP-2 variants havingamino acid substitutions at positions 8, 16, 24 and/or 28 of thewild-type GLP-2 sequence have increased biological activity compared to[Gly2]GLP-2 (SEQ ID NO:54) in C57BL mice.

Example 9 Dose-Response Effect of Selected Compounds on IntestinalGrowth in C57BL Mice

1820 (SEQ ID NO:10), 1855 (SEQ ID NO:43), 1846 (SEQ ID NO:34), 1858 (SEQID NO:46), 1849 (SEQ ID NO:37), 1848 (SEQ ID NO:36), and 1857 (SEQ IDNO:45) were selected as lead compounds since these compounds bothincreased small intestine mass relative to [Gly2]GLP-2 (SEQ ID NO:54)(Example 8) and had increased chemical stability relative to [Gly2]GLP-2(SEQ ID NO:54) under stressful conditions (Example 7). The dose-responseeffect of 1820 (SEQ ID NO:10), 1855 (SEQ ID NO:43), ZP1846 (SEQ IDNO:34), 1858 (SEQ ID NO:46), 1849 (SEQ ID NO:37), 1848 (SEQ ID NO:36),and 1857 (SEQ ID NO:45) on small intestinal mass was determined in maleC57BL mice. Individual groups (n=6) of mice were given 5, 15, 45, 135 or405 nmol/kg of each compound, s.c, twice daily for three consecutivedays. For comparison purposes other groups of animals were given eitherequimolar doses of [Gly2]GLP-2 (SEQ ID NO:54) or vehicle (phosphatebuffered saline, pH 7.4) in the same dosing regimen. Twenty-four hoursafter the last-dose of compound had been given the mice were sacrificedand the small intestine (from the pylorus to the cecum emptied andweighed to determine the effect on small intestinal mass.

Results

The effect of 1820 (SEQ ID NO:10), 1855 (SEQ ID NO:43), 1846 (SEQ IDNO:34), 1858 (SEQ ID NO:46), 1849 (SEQ ID NO:37), 1848 (SEQ ID NO:36),and 1857 (SEQ ID NO:45) on small intestine mass, relative to the effectof the reference compound, [Gly2]GLP-2 (SEQ ID NO:54) is shown in FIGS.1 to 5. At each of the doses tested the effect of [Gly₂]GLP-2 (SEQ IDNO:54) on small intestine mass is standardised at 100%. The intestinalgrowth effects of the compounds 1820 (SEQ ID NO:10), 1855 (SEQ IDNO:43), 1846 (SEQ ID NO:34), 1858 (SEQ ID NO:46), 1849 (SEQ ID NO:37),1848 (SEQ ID NO:36), and 1857 (SEQ ID NO:45) according to the inventionwere determined based on the ability of the peptides to dose-dependentlyincrease SI mass relative to the effect of equimolar doses of[Gly₂]GLP-2 (SEQ ID NO:54). Based on these findings we can conclude thatGLP-2 analogues containing 8 substitutions (G2, E3, T5, L10, A11, A16,A24, A28 with regard to GLP-2 1809 (SEQ ID NO:1)) give rise to asignificant increase in the weight of the small intestine compared tomice treated with [Gly2]GLP-2 (SEQ ID NO:54).

In particular, substitution of Asp3 for Glu and Asn16 for Ala and Gln28for Ala effect the increase in the weight of the small intestine in aselective manner compared to the colon (1839 (SEQ ID NO:27) or 1840 (SEQID NO:28) in comparison with 1809 (SEQ ID NO:1)). Thus, substitution ofthe three amino acids Asp3, Asn16 and Gln28 results in a selectiveincrease of the small intestine weight relative to the colon mass.

Furthermore, substitution of the Asp8 for Serine results in anadditional increase in the selectivity, thus resulting in asupplementary increase in the weight of the small intestine withoutsignificant effecting the weight of the colon (1818 (SEQ ID NO:8), 1820(SEQ ID NO:10), 1844 (SEQ ID NO:32), 1846 (SEQ ID NO:34), 1848 (SEQ IDNO:36), 1849 (SEQ ID NO:37), 1852 (SEQ ID NO:40), 1853 (SEQ ID NO:41),1855 (SEQ ID NO:43), 1858 (SEQ ID NO:46)).

FIGS. 1 to 4 show the results of experiments in which the dose responseeffect of exemplified compounds 1846 (SEQ ID NO:34), 1855 (SEQ IDNO:43), 1848 (SEQ ID NO:36), 1857 (SEQ ID NO:45), 1849 (SEQ ID NO:37) onthe SI-BW to colon-BW ratio is shown relative to reference compound[Gly₂]GLP-2 (SEQ ID NO:54). At each of the doses tested the effect of[Gly₂]GLP-2 (SEQ ID NO:54) on small intestine mass is standardised at100%. All of the exemplified compounds share modifications at positions8, 16 and/or 28 and show increased selectivity for causing growth of thesmall intestine relative to the colon.

Example 10 Dose Response Effect of 1846 (SEQ ID NO:34), 1848 (SEQ IDNO:36), 1855 (SEQ ID NO:43), and 1857 (SEQ ID NO:45) on Small IntestinalAtrophy in Mice Administered 5-FU

The rapid rate of proliferation of the small intestinal stem cells,makes them a susceptible target to the cytotoxic effects of thechemotherapeutic agents used in anti-cancer therapies. Consequently,clinical use of the chemotherapeutic agent 5-fluorouracil (5-FU) isfrequently associated with small intestinal injury (atrophy and diarrheain cancer patients. We have previously shown that i.p. administration of50 mg/kg 5-FU once daily for four days induces significant smallintestinal atrophy in C57BL mice. The effect of the lead compounds, 1846(SEQ ID NO:34), 1848 (SEQ ID NO:36), 1855 (SEQ ID NO:43), and 1857 (SEQID NO:45) on 5-FU-induced small intestinal atrophy was investigated inmice. We have previously shown that i.p. administration of 50 mg/kg 5-FUonce daily for four days induces significant small intestinal atrophy inC57BL mice. 1846 (SEQ ID NO:34), 1848 (SEQ ID NO:36), 1855 (SEQ IDNO:43), or 1857 (SEQ ID NO:45) were administered twice daily for threedays prior to 5-FU and for four days together with 5-FU administration.The lead compounds were each administered at five different doses (5,15, 45, 135 and 405 nmol/kg) that have been previously shown toeffectively stimulate small intestine growth in healthy mice (Example9). For comparison purposes a group of animals were treated with 405nmol/kg [Gly2]GLP-2 (SEQ ID NO:54). To determine the effect of 5-FU onthe small intestine a group of animals were given 5-FU I alone and leftuntreated (5-FU controls) and another group of animals were only givenvehicle (PBS controls).

Results

5-FU induced a significant decrease in SI-BW and SI length in C57BLmice, relative to PBS controls. The dose-response effect of 1846 (SEQ IDNO:34), 1848 (SEQ ID NO:36), 1855 (SEQ ID NO:43), or 1857 (SEQ ID NO:45)on SI-BW and SI length in mice administered 5-FU is shown in FIGS. 6 to9. The effect of 405 nmol/kg [Gly2]GLP-2 (SEQ ID NO:54) is also shown.1846 (SEQ ID NO:34), 1848 (SEQ ID NO:36), 1855 (SEQ ID NO:43), or 1857(SEQ ID NO:45) dose-dependently prevented 5-FU-induced SI atrophy andmaintained SI-BW at levels similar to PBS controls. ZP1848 (SEQ IDNO:36), ZP1855 (SEQ ID NO:43), and ZP1857 (SEQ ID NO:45), given at anequimolar dose, were significantly more efficacious than 405 nmol/kg[Gly2]GLP-2 (SEQ ID NO:54) on SI-BW. 1848 (SEQ ID NO:36) and 1857 (SEQID NO:45), were significantly more efficacious than 405 nmol/kg[Gly2]GLP-2 (SEQ ID NO:54) on SI-length.

Example 11 Dose Response Effect of 1846 (SEQ ID NO:34) on SmallIntestinal Atrophy and Diarrhea in SD Rats Administered 5-FU

The effect of the clinical candidate, 1846 (SEQ ID NO:34), on5-FU-induced small intestinal atrophy and diarrhea was investigated inSD rats. We have previously shown that i.p. administration of 75 mg/kg5-FU once daily for four days induces significant small intestinalatrophy and diarrhea in SD rats. 1846 (SEQ ID NO:34) (16, 80 and 400nmol/kg/d; n=20 rats/dose group) was administered twice daily for threedays prior to 5-FU and for four days together with 5-FU administration.5-FU controls and PBS controls were included in the study. Twenty-fourhours after the last dose of 1846 (SEQ ID NO:34) was given a subset ofanimals were sacrificed to determine the effect of 1846 (SEQ ID NO:34)on small intestinal atrophy. To determine the effect of 1846 (SEQ IDNO:34) on diarrhea all the animals were observed twice daily (morningand evening), during the dosing period and for an additional six days.At each observation period each animal was given a score (0, nodiarrhea, 1 (mild), fecal staining around the anus, 2 (moderate), fecalstaining on the hind limbs and tail and 3 (severe) fecal staining on thefront limbs and abdomen) that indicated whether or not the animal haddiarrhea and the severity of the diarrhea

Results

5-FU induced a significant decrease in SI-BW and SI length and induceddiarrhea, in SD rats, relative to PBS controls. The dose-response effectof 1846 (SEQ ID NO:34) on 5-FU induced small intestinal atrophy anddiarrhea is shown in FIGS. 10 and 11. 1846 (SEQ ID NO:34)dose-dependently prevented 5-FU induced small intestinal atrophy andmaintained SI-BW and SI length at levels similar to the controls. At thehighest dose (400 nmol/kg) administered, 1846 (SEQ ID NO:34), decreasedthe incidence and severity of diarrhea in rats administered 5-FU

Example 12 Dose Response Effect of 1846 (SEQ ID NO:34) on Crypt-VillusLength and Muscularis Thickness in the Small Intestine of SD Rats

The effect of the clinical candidate, 1846 (SEQ ID NO:34), oncrypt-villus length and muscularis thickness in the small intestine ofSD rats was investigated. 1846 (SEQ ID NO:34) (0.62, 3.41 or 6.2mg/kg/day, n=6 rats/dose group) was administered as an i.v. bolus, oncedaily for five consecutive days. Twenty-four hours after the last dosehad been administered the rats were sacrificed and a 1 cm biopsy wasexcised from the jejunum (30 cm distal from the gastric duodenaljunction) and from the ileum (30 cm proximal from the ileocaecaljunction) for histological processing.

Results

The dose-response effect of 1846 (SEQ ID NO:34) on crypt-villus lengthand muscularis thickness in the jejunum and ileum is shown in FIG. 12.1846 (SEQ ID NO:34) dose-dependently increased mean crypt-villus lengthin the jejunum and ileum muscularis thickness in the ileum.

Example 13 Effect of 1848 (SEQ ID NO:36) on Markers of Small IntestinalInflammation in an Indomethacin-Induced Model of Crohn's Disease

Crohn's disease is a chronic disease that causes episodic inflammationof the small intestine. The effect of the GLP-2 analogue 1848 (SEQ IDNO:36) on small intestinal inflammation in an indomethacin-induced modelof Crohn's disease was investigated. We have previously shown thatadministration of indomethacin (s.c, once daily for 2 days) inducessmall intestinal inflammation characterized by ulcerations and,increases in the pro-inflammatory cytokine, tumour necrosis factor alpha(TNF-α).

To determine the effect of ZP1848 (SEQ ID NO:36) on ulceration 1848 (SEQID NO:36) (8, 40 and 200 nmol/kg, s.c, twice daily (9:00 and 16:00)) wasgiven for 4 days prior to the first dose of indomethacin and for anadditional two days together with indomethacin. The corticosteroid,prednisolone (10 mg/kg, p.o) was used as a positive control sincecorticosteroids are commonly used in the treatment of activeinflammation in Crohn's disease. In addition a group of animals weregiven both 1848 (SEQ ID NO:36) (200 nmol/kg) and prednisolone todetermine the effects of a combination treatment. Twenty four hoursafter the last dose of 1848 (SEQ ID NO:36) had been given the animalswere sacrificed the small intestine was gently flushed clean and fixed.To determine the extent of ulceration the intestine was cut open alongthe antimesenteric margin, suspended on a polypropylene plate andsurface-stained with Alcian Green 3BX. Starting at the pylorus, thesmall intestine was scanned and the shape (circular vs. linear) and size(circular ulcers: diameter, linear ulcers: length×width) of all ulcerswas measured using a standard ruler (resolution: 0.5 mm). An ulcer wasdefined as an area, which lacked epithelial surface. Finally the totaldamaged area was calculated for each animal by summation of the areas ofall individual ulcers.

To determine the effect of 1848 (SEQ ID NO:36) on the concentrations ofTNF-α in the small intestine 1848 (SEQ ID NO:36) and Indomethacin weregiven as described above. At sacrifice, however the small intestine wasseparated into three segments of equal length (proximal, mid anddistal). TNF-α concentrations were measured in each of the individualsegments using a commercially available ELISA kit.

Results

Indomethacin caused a strong induction of small intestinal ulcers,compared to the control group ((estimated extent of ulceration 333±21mm² vs. 10 mm². The effect of 1846 (SEQ ID NO:34) on the estimatedextent of ulceration (mm²) is shown in FIG. 13. Treatment with 1848 (SEQID NO:36) (8 nmol/kg, 40 nmol/kg and 200 nmol/kg, significantlydecreased the extent of ulceration (230±12 mm², 216±17 mm², and 178±17mm² respectively, p<0.001 vs. indomethacin). At the highest dose (200nmol/kg) used ZP1848 (SEQ ID NO:36) was more effective than the positivecontrol, prednisolone (p<0.05).

Indomethacin caused an approx. 2.9-fold increase in tissue levels ofTNF-α in the proximal segment (97±14 pg/mg protein) compared to controlanimals (34±7 pg/mg protein, p<0.05 vs. indomethacin). The effect of1846 (SEQ ID NO:34) on small intestinal TNF-α concentrations is shown inFIG. 14. Treatment with 1848 (SEQ ID NO:36) (8, 40 or 200 nmol/kg),significantly reduced tissue levels of TNF-α (45±14 pg/mg protein, 44±9pg/mg protein and 45±7 pg/mg protein, respectively) with no significantdifference between the 3 doses.

Indomethacin caused an approx. 3.2-fold increase in tissue levels ofTNF-α in the mid segment (108±9 pg/mg protein) compared to controlanimals (34±6 pg/mg protein, p<0.05 vs. indomethacin). 1848 (SEQ IDNO:36) (40 or 200 nmol/kg) significantly reduced tissue levels of TNF-αrespectively, p<0.05 vs. indomethacin)

Indomethacin caused an approx. 1.7-fold increase in tissue levels ofTNF-α in the distal segment (75±5 pg/mg protein) compared to controlanimals (45±3 pg/mg protein, p<0.05 vs. indomethacin). 1848 (SEQ IDNO:36) had an inhibitory effect on TNF-α levels in the distal segmentbut the effect was less pronounced compared to the other segments.Prednisolone alone did not significantly affect tissue levels of TNF-αin all 3 segments but prednisolone administration improved theinhibitory effect of 1848 (SEQ ID NO:36) (200 nmol/kg) on TNF-α levelsexclusively in the distal segment.

Example 14

Formulation of ZP1846 (SEQ ID NO: 34), 10 Mg/ml in Histidine, Mannitoland Acetate Formulation

-   1. Fill 800 mL (WFI) water in to a 1 L beaker-   2. Weigh 13.964 g L-histidine in a beaker and add to the 1 L beaker-   3. Weigh 32.200 g Mannitol in a beaker and add to the 1 L beaker-   4. Add 629 μL 100% acetic acid directly in to the 1 L beaker or    weigh 12.58 g of a 5% (w/v) acetic acid solution and add to the    beaker.-   6. Fill to approximately 950 mL-   7. Measure pH and adjust to pH 6.9-7.0 with 10% Acetic acid or 0.25    M Histidine if necessary-   8. Weigh 11.312 g Drug Substance (peptide content 88.4%) and add to    the beaker-   9. Fill to 1.015 kg (=approximately 1000 mL) and measure pH,    osmolarity and density.-   10. Sterile filter the formulation through two sterile filters    connected in series.-   11. Dispense the formulation in 0.5 mL aliquots in a LAF bench into    2 mL pharmaceutically approved vials.-   12. Partially place freeze-drying stoppers before loading into a    lyophilizer that has been sterilized and pre-cooled to 4° C.-   13. A lyophilization cycle is run over 40.5 hours consisting of    freezing, annealing, primary drying and secondary drying phases. The    vials are stoppered under nitrogen while in the lyophilizer chamber.-   14. The vials are sorted and overseals and crimps are applied.

Example 15

Formulation of ZP1846 (SEQ ID NO:34), 10 Mg/ml in Histidine, Arginine,Mannitol and Trehalose

-   1. Fill 800 mL (WFI) water in to a 1 L beaker-   2. Weigh 6.206 g L-Histidine in a beaker and add to the 1 L beaker-   3. Weigh 3.484 g L-Arginine in a beaker and add to the 1 L beaker-   4. Weigh 33.46 g Mannitol in a beaker and add to the 1 L beaker-   5. Weigh 11.16 g Trehalose in a beaker and add to the 1 L beaker-   6. Fill to approximately 950 mL-   7. Measure pH and adjust to pH 6.9-7.0 with 10% Acetic acid or 0.25    M Histidine if necessary-   8. Weigh 11.312 g Drug Substance (peptide content 88.4%) and add to    the beaker-   9. Fill to 1.015 kg (=approximately 1000 mL) and measure pH,    osmolarity and density.-   10. Sterile filter the formulation through two sterile filters    connected in series.-   11. Dispense the formulation in 0.5 mL aliquots in a LAF bench into    2 mL pharmaceutically approved vials.-   12. Partially place freeze-drying stoppers before loading into a    lyophilizer that has been sterilized and pre-cooled to 40° C.-   13. A lyophilization cycle is run over 40.5 hours consisting of    freezing, annealing, primary drying and secondary drying phases. The    vials are stoppered under nitrogen while in the lyophilizer chamber.-   14. The vials are sorted and overseals and crimps are applied

While the invention has been described in conjunction with the exemplaryembodiments described above, many equivalent modifications andvariations will be apparent to those skilled in the art when given thisdisclosure. Accordingly, the exemplary embodiments of the invention setforth are considered to be illustrative and not limiting. Variouschanges to the described embodiments may be made without departing fromthe spirit and scope of the invention. All documents including patents,patent applications, and publications cited herein are expresslyincorporated by reference.

1. A glucagon-like peptide-2 (GLP-2) analog wherein said analog has theamino acid sequence: H-HGEGSFSSELSTILDALAARDFIAWLIATKITDK₆-NH₂ (SEQ IDNO:34), or a pharmaceutically acceptable salt thereof.
 2. Apharmaceutical composition comprising: (a) the GLP-2 analog of claim 1,or a pharmaceutically acceptable salt thereof; and (b) apharmaceutically acceptable carrier.